Immunoneutral silk-fiber-based medical devices

ABSTRACT

Silk is purified to eliminate immunogenic components (particularly sericin) and is used to form fabric that is used to form tissue-supporting prosthetic devices for implantation. The fabrics can carry functional groups, drugs, and other biological reagents. Applications include hernia repair, tissue wall reconstruction, and organ support, such as bladder slings. The silk fibers are arranged in parallel and, optionally, intertwined (e.g., twisted) to form a construct; sericin may be extracted at any point during the formation of the fabric, leaving a construct of silk fibroin fibers having excellent tensile strength and other mechanical properties.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.10/008,924, filed Nov. 16, 2001, the entire teachings of which areincorporated herein by reference. Priority to provisional applicationU.S.S.No. 60/453,584, filed Mar. 11, 2003, is also claimed, and itsentire contents are also incorporated by reference herein.

BACKGROUND

[0002] Disease, aging, trauma or chronic wear often lead to tissue ororgan failure. In treating such failures, the goal of many clinicalprocedure is restoration of function. A patient often requiresadditional support, beyond the body's own means of healing, such assurgery or the implantation of a medical device. Such procedures areoften needed to combat permanent disability and even death. The fieldsof biomaterials and tissue engineering are providing new options togradually restore native tissue and organ function through the researchand development of temporary scaffolds, matrices, and constructs (i.e.,devices) that initially support a disabled tissue or organ, buteventually allow for the development and remodeling of the body's ownbiologically and mechanically functional tissue.

[0003] The responsibilities or design requirements of such a scaffoldinclude: (i) the ability to provide immediate mechanical stabilizationto the damaged or diseased tissue, (ii) support cell and tissue ingrowthinto the device, (iii) communicate the mechanical environment of thebody to the developing tissue; such is achieved through the propermechanical and biological design of the device, (iv) degrade at such arate that the ingrowing cells and tissue have sufficient time toremodel, thus creating new autologous function tissue that can survivethe life of the patient. In certain instances, the device should mimicthe correct three-dimensional structure (e.g., a bone scaffold) of thetissue it is attempting to support. In other instances, the device mayserve as a temporary ligature (e.g., a flat mesh for hernia repair or ahemostat for bleeding) to a three-dimensional tissue (abdominal wallmuscle in the case of hernia). Regardless of application, the presentdirection of the medical device field is the complete restoration ofbodily function through the support of autologous tissue development.

[0004] Unfortunately, most biomaterials-available today do not possessthe mechanical integrity of high load demand applications (e.g., bone,ligaments, tendons, muscle) or the appropriate biological functionality;most biomaterials either degrade too rapidly (e.g., collagen, PLA, PGA,or related copolymers) or are non-degradable (e.g., polyesters, metal),where in either case, functional autologous tissue fails to develop andthe patient suffers disability. In certain instances a biomaterial maymisdirect tissue differentiation and development (e.g., spontaneous boneformation, tumors) because it lacks biocompatibility with surroundingcells and tissue. As well, a biomaterial that fails to degrade typicallyis associated with chronic inflammation, where such a response isactually detrimental to (i.e., weakens) surrounding tissue.

[0005] If properly designed, silk may offer new clinical options for thedesign of a new class of medical devices, scaffolds and matrices. Silkhas been shown to have the highest strength of any natural fiber, andrivals the mechanical properties of synthetic high performance fibers.Silks are also stable at high physiological temperatures and in a widerange of pH, and are insoluble in most aqueous and organic solvents.Silk is a protein, rather than a synthetic polymer, and degradationproducts (e.g., peptides, amino acids) are biocompatible. Silk isnon-mammalian derived and carries far less bioburden than othercomparable natural biomaterials (e.g., bovine or porcine derivedcollagen).

[0006] Silk, as the term is generally known in the art, means afilamentous fiber product secreted by an organism such as a silkworm orspider. Silks produced from insects, namely (i) Bombyx mori silkworms,and (ii) the glands of spiders, typically Nephilia clavipes, are themost often studied forms of the material; however, hundreds to thousandsof natural variants of silk exist in nature. Fibroin is produced andsecreted by a silkworm's two silk glands. As fibroin leaves the glands,it is coated with sericin, a glue-like substance. However, spider silkis valued (and differentiated from silkworm silk) as it is produced as asingle filament lacking any immunogenic contaminates, such as sericin.

[0007] Unfortunately, spider silk can not be mass produced due to theinability to domesticate spiders; however, spider silk, as well as othersilks can be cloned and recombinantly produced, but with extremelyvarying results. Often, these processes introduce bioburdens, arecostly, cannot yield material in significant quantities, result inhighly variable material properties, and are neither tightly controllednor reproducible.

[0008] As a result, only silkworm silk has been used in biomedicalapplications for over 1,000 years. The Bombyx mori specie of silkwormproduces a silk-fiber (known as a “bave”) and uses the fiber to buildits cocoon. The bave, as produced, includes two fibroin filaments or“broins”, which are surrounded with a coating of gum, known assericin-the silk fibroin filament possesses significant mechanicalintegrity. When silk fibers are harvested for producing yarns ortextiles, including sutures, a plurality of fibers can be alignedtogether, and the sericin is partially dissolved and then resolidifiedto create a larger silk fiber structure having more than two broinsmutually embedded in a sericin coating.

[0009] As used herein, “fibroin” includes silkworm fibroin (i.e. fromBombyx mori) and fibroin-like fibers obtained from spiders (i.e. fromNephila clavipes). Alternatively, silk protein suitable for use in thepresent invention can be obtained from a solution containing agenetically engineered silk, such as from bacteria, yeast, mammaliancells, transgenic animals or transgenic plants. See, for example, WO97/08315 and US Pat. No. 5,245,012.

[0010] Silkworm silk fibers, traditionally available on the commercialmarket for textile and suture applications are often “degummed” andconsist of multiple broins plied together to form a larger singlemulti-filament fiber. Degumming here refers to the loosening of thesericin coat surrounding the two broins through washing or extraction inhot soapy water. Such loosening allows for the plying of broins tocreate larger multifilament single fibers. However, complete extractionis often neither attained nor desired. Degummed silk often contains oris recoated with sericin and/or sericin impurities are introduced duringplying in order to congeal the multifilament single fiber. The sericincoat protects the frail fibroin filaments (only ˜5 microns in diameter)from fraying during traditional textile applications wherehigh-through-put processing is required. Therefore, degummed silk,unless explicitly stated as sericin-free, typically contain 10-26% (byweight) sericin (see Tables 1 & 2).

[0011] When typically referring to “silk” in the literature, it isinferred that the remarks are focused to the naturally-occurring andonly available “silk” (i.e., sericin-coated fibroin fibers) which havebeen used for centuries in textiles and medicine. Medical grade silkwormsilk is traditionally used in only two forms: (i) as virgin silk suture,where the sericin has not been removed, and (ii) the traditional morepopular silk suture, or commonly referred to as black braided silksuture, where the sericin has been completely removed, but replaced witha wax or silicone coating to provide a barrier between the silk fibroinand the body tissue and cells. Presently, the only medical applicationfor which silk is still used is in suture ligation, particularly becausesilk is still valued for it mechanical properties in surgery (e.g., knotstrength and handlability).

[0012] Despite virgin silk's use as a suture material for thousands ofyears, the advent of new biomaterials (collagen, synthetics) haveallowed for comparisons between materials and have identified problemswith sericin. Silk, or more clearly defined as Bombyx mori silkwormsilk, is non-biocompatible. Sericin is antigenic and elicits a strongimmune, allergic or hyper-T-cell type (versus the normal mild “foreignbody” response) response. Sericin may be removed (washed/extracted) fromsilk fibroin; however, removal of sericin from silk changes theultrastructure of the fibroin fibers, exposing them, and results in lossof mechanical strength, leading to a fragile structure.

[0013] Extracted silk structures (i.e., yarns, matrices) are especiallysusceptible to fraying and mechanical failure during standard textileprocedures due to the multifilament nature of the smaller diameter (˜5um) fibroin filaments. The extracted fibroin's fragility is the reasonthat when using silk in the design and development of medical devices,following extraction, it is typically taught (Perez-Rigueiro, J. AppI.Polymer Science, 70, 2439-2447, 1998) that you must dissolve andreconstitute silk using standard methods (U.S. Pat. No. 5,252,285) togain a workable biomaterial. The inability to handle extracted silkfibroin with present-day textile methods and machinery has prevented theuse of non-dissolved sericin-free fibroin from being explored as amedical device.

[0014] Additional limitations of silk fibroin, whether extracted fromsilkworm silk, dissolved and reconstituted, or produced from spiders orinsects other than silkworms include (i) the hydrophobic nature of silk,a direct result of the beta-sheet crystal conformation of the corefibroin protein which gives silk its strength, (ii) the lack of cellbinding domains typically found in mammalian extracellular matrixproteins (e.g., the peptide sequence RGD), and (iii) silk fibroin'ssmooth surface. As a result, cells (e.g., macrophages, neutrophils)associated with an inflammatory and host tissue response are unable torecognize the silk fibroin as a material capable of degradation. Thesecells thus opt to encapsulate and wall off the foreign body (see FIG.18A) thereby limiting (i) silk fibroin degradation, (ii) tissueingrowth, and (iii) tissue remodeling. Thus, silk fibroin filamentsfrequently induce a strong foreign body response (FBR) that isassociated with chronic inflammation, a peripheral granuloma and scarencapsulation (FIG. 18A).

[0015] In addition to the biological disadvantages of silk, themultifilament nature of silk (e.g., as sutures) as well as the smallsize of the fibroin filaments can lead to a tightly packed structure. Assuch, silk may degrade too rapidly. Proteases (enzymes) produced fromthe stimulated cells found within the peripheral encapsulation canpenetrate the implanted structure (see FIG. 11A and FIG. 11B), but cellsdepositing new tissue (e.g., fibroblasts) which may reinforce the device(in this case a black braided suture) during normal tissue remodelingcannot. Therefore, the interior of non-treated or non-modified fibroindevices does not come in contact with the host foreign body response andtissue (led and produced by fibroblasts) and as a result, the capacityof the device to direct tissue remodeling is limited. Host cell andtissue growth is limited and degradation is not normally possible.

[0016] In the case of sutures, it is thought that these problems can bemanaged by treating fibroin sutures with cross-linking agents or bycoating the sutures with wax, silicone or synthetic polymers, therebyshielding the material from the body. Coatings, such as sericin, wax orsilicone, designed to add mechanical stability to the fibroin (combatingits fragility while providing a barrier between the body and thefibroin), limits cell attachment, recognition and infiltration andtissue ingrowth and fibroin degradation. As a result, silk istraditionally thought of as a non-degradable material.

[0017] Classification as a non-degradable may be desirable when silk isintended for use as a traditional suture ligation device, i.e., cell andtissue ingrowth into the device are not desirable. Therefore, cellattachment and ingrowth (which lead to matrix degradation and activetissue remodeling) is traditionally prevented by both the biologicalnature of silk and the structure's mechanical design. In fact, a generalbelief that silk must be shielded from the immune system and theperception that silk is non-biodegradeable have limited silk's use insurgery. Even in the field of sutures, silk has been displaced in mostapplications by synthetic materials, whether biodegradable or permanent.

[0018] Therefore, there exists a need to generate sericin-extractedsilkworm fibroin fibers that are biocompatible, promote ingrowth ofcells, and are biodegradable.

SUMMARY

[0019] Natural silk fibroin fiber constructs, disclosed herein, offer acombination of high strength, extended fatigue life, and stiffness andelongation at break properties that closely match those of biologicaltissues. The fibers in the construct are non-randomly aligned into oneor more yarns. The fiber constructs are biocompatible (due to theextraction of sericin from the silkworm silk fibers) and substantiallyfree of sericin. The fiber constructs are further non-immunogenic; i.e.,they do not elicit a substantial allergic, antigenic, or hyper T-cellresponse from the host, diminishing the injurious effect on surroundingbiological tissues, such as those that can accompany immune-systemresponses in other contexts. In addition, the fiber constructs promotethe ingrowth of cells around said fibroin fibers and are biodegradable.

[0020] Indications that the fiber construct is “substantially free” ofsericin mean that sericin comprises less than 20% sericin by weight.Preferably, sericin comprises less than 10% sericin by weight. Mostpreferably, sericin comprises less than 1% sericin by weight (see Table2). Furthermore, “substantially free” of sericin can be functionallydefined as a sericin content that does not elicit a substantialallergic, antigenic, or hyper T-cell response from the host. Likewise,indication that there is less than a 3% change in mass after a secondextraction would imply that the first extraction “substantially removed”sericin from the construct and that the resulting construct was“substantially free” of sericin following the first extraction (seeTable 2 and FIG. 1F).

[0021] Methods of this disclosure extract sericin from the constructmuch more thoroughly than do the typical “degumming” procedures thatcharacterize traditional processing practices for the production of silktextiles for non-surgical applications (see above for definition). FIG.1A shows an image of a degummed fiber where fibroin filaments were pliedtogether forming a larger fiber re-encased with sericin. This “degummed”fiber contains ˜26%, by weight, sericin. In a preferred embodiment, thesericin-extracted silkworm fibroin fibers retain their native proteinstructure and have not been dissolved and reconstituted.

[0022] “Natural” silk fibroin fibers are produced by an insect, such asa silkworm or a spider and possess their native, as formed, proteinstructure. Preferably, the silk fibroin fiber constructs arenon-recombinant (i.e., not genetically engineered) and have not beendissolved and reconstituted. In a preferred embodiment, thesericin-extracted fibroin fibers comprised fibroin fibers obtained fromBombyx mori silkworm. Further, the term, “biodegradable,” is used hereinto mean that the fibers are degraded within one year when in continuouscontact with a bodily tissue. In addition, our data suggests (FIG. 13A-E, FIG. 18 A-C & FIG. 19 A-D) that the rate of degradation can beinfluenced and enhanced by surface modification of the fibroin (FIG.13A-D & FIG. 18A-C) as well as the geometric configuration of the yarnand/or fabric (FIG. 19A-D). In one embodiment, silk fibroin yarn lost50% of its ultimate tensile strength within two weeks followingimplantation in vivo (FIG. 12) and 50% of its mass within approximately30 to 90 days in vivo, depending on implantation sight (FIG. 13A-D). Thechoice of implantation site in vivo (e.g., intra-muscular versussubcutaneous) was shown to significantly influence the rate ofdegradation (FIG. 13A-D).

[0023] Textile-grade silk” is naturally occurring silk that includes asericin coating of greater than 19%-28% by weight of the fiber. “Suturesilk” is silk that either contains sericin (“virgin silk suture”) or iscoated with a hydrophobic composition, such as bee's wax, paraffin wax,silicon, or a synthetic polymeric coating (“black braided silk suture”).The hydrophobic composition repels cells or inhibits cells fromattaching to the coated fiber. Black braided silk is a suture silk inwhich sericin has been extracted and replaced with additional coating.Suture silk is typically non-biodegradable.

[0024] Due to the absence of a protective wax or other hydrophobiccoating on the fibers the silk fibroin constructs described arebiologically (coupling of cell binding domains) and/or mechanically(increase silk surface area and decrease packing density) designed topromote increased cell infiltration compared to textile-grade silk orsuture silk when implanted in bodily tissue. As a result, the silkfibroin constructs support cell ingrowth/infiltration and improved cellattachment and spreading, which leads to the degradation of the silkfibroin construct thereby essentially creating a new biodegradablebiomaterial for use in medical device and tissue engineeringapplications. The ability of the fiber construct to support cellattachment and cell and tissue ingrowth/infiltration into the construct,which in return supports degradation, may be further enhanced throughfibroin surface modification (peptide coupling using RGD, chemicalspecies modification and increasing hydrophilicity through gas plasmatreatment) and/or the mechanical design of the construct therebyincreasing material surface area thus increasing its susceptibility tothose cells and enzymes that possess the ability to degrade silk. Thesilk fibers are optionally coated with a hydrophilic composition, e.g.,collagen or a peptide composition, or mechanically combined with abiomaterial that supports cell and tissue ingrowth to form a compositestructure. The choice of biomaterial, amount and mechanical interaction(e.g., wrapped or braided about a core of silk fibroin) can be used toalter and/or improve rates of cell ingrowth and construct degradation.

[0025] Fibers in the construct are non-randomly aligned with one anotherinto one or more yarns. Such a structure can be in a parallel, braided,textured, or helically-organized (twisted, cabled (e.g., a wire-rope))arrangement to form a yarn. A yarn may be defined as consisting of atleast one fibroin fiber. Preferably, a yarn consists of at least threealigned fibroin fibers. A yarn is an assembly of fibers twisted orotherwise held together in a continuous strand. An almost infinitenumber of yarns may be generated through the various means of producingand combining fibers. A silk fiber is described above; however, the termfiber is a generic term indicating that the structure has a length 100times greater than its diameter.

[0026] When the fibers are twisted or otherwise intertwined to form ayarn, they are twisted/intertwined enough to essentially lock in therelative fiber positions and remove slack but not so much as toplastically deform the fibers (i.e., does not exceed the material'syield point), which compromises their fatigue life (i.e., reduces thenumber of stress cycles before failure). The sericin-free fibroin fiberconstructs can have a dry ultimate tensile strength (UTS) of at least0.52 N/fiber (Table 1, 4), and a stiffness between about 0.27 and about.0.5 N/mm per fiber. Depending on fiber organization and hierarchy, wehave shown that fibroin construct UTSs can range from 0.52 N/fiber toabout 0.9N/fiber. Fibroin constructs described here retained about 80%of their dry UTS and about 38% of their dry stiffness, when tested wet(Table 5). Elongations at-break between about 10% and about 50% weretypical for fibroin constructs tested in both dry and wet states.Fibroin constructs typically yielded at about 40 to 50% of their UTS andhad a fatigue life of at least 1 million cycles at a load of about 20%of the yarns ultimate tensile strength.

[0027] In one embodiment of the present invention, the alignedsericin-extracted silkworm fibroin fibers are twisted about each otherat 0 to 11.8 twists per cm (see Table 6 & 7).

[0028] The number of hierarchies in the geometrical structure of thefiber construct as well as the number offibers/groups/bundles/strands/cords within a hierarchical level, themanner of intertwining at the different levels, the number of levels andthe number of fibers in each level can all be varied to change themechanical properties of the fiber construct (i.e., yarn) and therefore,fabric (Table 4 & 8). In one embodiment of the present invention, thefiber construct (i.e. yarn) is organized in a single-level hierarchicalorganization, said single-level hierarchical organization comprising agroup of parallel or intertwined yarns. Alternatively, the fiberconstruct (i.e. yarn) organized in a two-level hierarchicalorganization, said two-level hierarchical organization comprising abundle of intertwined groups. In another embodiment of the presentinvention, the fiber construct (i.e. yarn) is organized into athree-level hierarchical organization, said three-level hierarchicalorganization comprising a strand of intertwined bundles. Finally,another embodiment of the present invention, the fiber construct (i.e.yarn) is organized into a four-level hierarchical organization, saidfour-level hierarchical organization comprising a cord of intertwinedstrands.

[0029] The sericin can be removed from the fibroin fibers before thealignment into a yarn or at a higher level in the hierarchical geometryof the fiber construct. The yarn is handled at low tension (i.e., theforce applied to the construct will never exceed the material's yieldpoint during any processing step) and with general care and gentlenessafter the sericin is removed. Processing equipment is likewiseconfigured to reduce abrasiveness and sharp angles in the guide fixturesthat contact and direct the yarn during processing to protect thefragile fibroin fibers from damage; extraction residence times of 1 hourare sufficient to extract sericin but slow enough as not to damage theexposed filaments. Interestingly, when a silk fiber construct consistingof multiple fibers organized in parallel has been extracted under theseconditions, a “single” larger sericin free yarn resulted (i.e.,individual fibers cannot be separated back out of the construct due tothe mechanical interaction between the smaller fibroin filaments onceexposed during extraction). Furthermore, as a result of the mechanicalinterplay between the sericin-free micro filaments, extraction oftwisted or cabled yarns has typically resulted in less “lively” yarnsand structures. As a result of this phenomenon, a greater degree offlexibility existed in the design of the yarns and resulting fabrics;for example, higher twist per inch (TPI) levels can be used, which wouldnormally create lively yarns that would be difficult to form intofabrics. The added benefit of higher TPIs was the reduction in yarn andfabric stiffness. (i.e. matrix elasticity can be increased)(Tables 6 and7; FIG. 6A and FIG. 6B).

[0030] A plurality of yarns are intertwined to form a fabric. Fabricsare generated through the uniting of one or more individual yarnswhereby the individual yarns are transformed into textile and medicaldevice fabrics. In one embodiment of the present invention, the yarn istwisted at or below 30 twists per inch. Fabrics are produced or formedby non-randomly combining yarns: weaving, knitting, or stitch bonding toproduce completed fabrics. In one embodiment, this combining of yarns toform a fabric is done on a machine. However, it is very important tonote that the end fabric product is distinct based on the yarn type usedto make it thus providing tremendous power through yarn design to meetclinical needs. A fabric can be, but is not limited to, woven, knit,warp-knit, bonded, coated, dobby, laminated, mesh, or combinationsthereof.

[0031] Of note, the textile methods of braiding, in addition to makingyarns, can also be used to make fabrics, such as a flat braided fabricor a larger circular braid (FIG. 4A). Inversely, weaving and knitting,two fabric forming methods, although not commonly used, can also be usedto make yarns. In such instances, the differentiation between a “yarn”and a “fabric” is not entirely apparent, and the homogeneity should beused to make clear distinctions, i.e., a yarn is typically morehomogeneous in composition and structure than a fabric.

[0032] In one embodiment of the present invention, multiple silkwormsilk fibers may be organized helically (e.g., twisted or cabled) or inparallel, in a single hierarchical level or in multiple levels,extracted, and used to create a braided suture for tissue ligation. Inanother embodiment, the mechanical interaction of extracted fibroinfilaments in a twisted or cabled configuration following extraction canbe used as a medical suture.

[0033] Non-woven fabrics may be formed by randomly organizing aplurality of yarns, or a single yarn cut into many small length pieces.Non-limiting examples include a fabric for hemostat or bone scaffold.All fabrics can either derive from a single yarn construct (homogenous)or multiple yarns constructs (heterogeneous). The ability to design fora variety of silk fibroin yarn structures, as described in detail below,dramatically increases fabric design potential when considering aheterogeneous fabric structure.

[0034] In one embodiment of the present invention, the fabric is acomposite of the sericin-extracted fibroin fibers or yarns and one ormore degradable polymers selected from the group consisting ofCollagens, Polylactic acid or its copolymers, Polyglycolic acid or itscopolymers, Polyanhydrides, Elastin, Glycosamino glyccands, andPolysaccharides. Furthermore, the fabric of the present invention may bemodified to comprise a drug associated or a cell-attachment factorassociated with fabric (i.e. RGD). In one embodiment of the presentinvention, the fabric is treated with gas plasma or seeded withbiological cells.

[0035] Additional aspects of this disclosure relate to the repair ofspecific bodily tissues, such as hernia repair, urinary bladder tissuesand slings, pelvic floor reconstruction, peritoneal wall tissues,vessels (e.g., arteries), muscle tissue (abdominal smooth muscle,cardiac), hemostats, and ligaments and tendons of the knee and/orshoulder as well as other frequently damaged structures due to trauma orchronic wear. Examples of ligaments or tendons that can be producedinclude anterior cruciate ligaments, posterior cruciate ligaments,rotator cuff tendons, medial collateral ligaments of the elbow and knee,flexor tendons of the hand, lateral ligaments of the ankle and tendonsand ligaments of the jaw or temporomandibular joint. Other tissues thatmay be produced by methods of this disclosure include cartilage (botharticular and meniscal), bone, skin, blood vessels, stents for vesselsupport and/or repair, and general soft-connective tissue.

[0036] In other aspects, silkworm fibroin fibers, in the form of a yarnor of a larger construct of yarns, now termed a device, is stripped ofsericin, and made (e.g., woven, knitted, non-woven wet laid, braided,stitch bonded, etc.) into a fabric, sterilized and used as animplantable supporting or repair material that offers a controllablelifetime (i.e., degradation rate) and a controllable degree of collagenand/or extracellular matrix deposition. The support or repair materialcan be used for any such purpose in the body, and in particular can beused for hernia repair, reconstruction of body walls, particularly inthe thorax and abdominal cavity, and support, positioning orimmobilization of internal organs, including, without limitation, thebladder, the uterus, the intestines, the urethra, and ureters.Alternatively, silkworm fibroin fibers may be stripped of sericin andorganized into a non-woven fabric. Such non-woven fabric can be used asan implantable supporting or repair material as above, but morespecifically for applications where a sponge formation would be useful.

[0037] The purified silk can be purified by any of a variety oftreatments that remove the sericin proteins found in the native fibrils.Sericin has been removed sufficiently when implants of purified silkelicit only a mild, transient foreign body reaction in the absecense ofan antigenic (B-cell, T-cell) response, i.e., are biocompatible. Aforeign body reaction is characterized by an inner layer of macrophagesand/or giant cells with a secondary zone of fibroblasts and connectivetissue. The degree of foreign body response has been shown to becontrollable through fibroin modification (FIG. 13A-D & FIG. 18A-C) andyarn design (FIG. 19A-D). Sericin can be removed from individualsilkworm fibroin fibers, a group of silkworm fibroin fibers (i.e. ayarn), having an organized orientation (e.g., parallel or twisted), orform a fabric or other construct comprising a plurality of yarns. Theconstruct can then be sterilized and implanted in an organism as amedical device.

[0038] Other features and advantages of the invention will be apparentfrom the following description of preferred embodiments thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0039]FIG. 1A is a scanning electron microscopy (SEM) image of a singlenative degummed and plied 20/22 denier silk fiber having a sericincoating.

[0040]FIG. 1B illustrates SEM of the silk fiber of FIG. 1A extracted for60 min at 37° C.

[0041]FIG. 1C illustrates SEM of the silk fiber of FIG. 1A extracted for60 min at 90° C. and illustrating complete removal of the sericincoating.

[0042]FIG. 1D is a chart showing ultimate tensile strength (UTS) andstiffness (N/mm for a 3 cm length matrix) as a function of extractionconditions.

[0043]FIG. 1E illustrates SEM of a raw silk fibroin. FIG. 1F illustratesa first extraction at 90° for 60 min. FIG. 1G illustrates a secondextraction under identical conditions. These figures show mechanicaldamage to the filaments that results in a typical 3% mass loss followingthe second extraction. Therefore, as long as the % mass loss does notchange more than 3% from the first to the second extraction (90° C., 1hr, standard detergent and salt), it is assumed that complete extractionhas been achieved. The utility of a 3% loss in total mass loss reflectsthe variability in the measurements, assays and mechanical damageresulting in mass loss of the yarn following the second extraction.

[0044]FIG. 2A is a representative 3-D model of a (cable or twisted) yarndepicting its 5 levels of hierarchy (single fiber level not shown).Depending on the number of fibers used in each level, the cord couldserve as either a yarn for knitting a hernia repair mesh or as a cord tobe used in parallel with other cords to form an ACL matrix.

[0045]FIG. 2B is a schematic depicting the generation of a two-levelhierarchical twisted or cabled yarn containing 36 fibers before beingplied in parallel to form an ACL matrix or used to generate a weave-orknit fabric for tissue engineering and tissue repair (e.g. hernia mesh).The schematic representations visually define two very popular forms offabric formations: a “weave” and a “knit.”

[0046]FIG. 2C illustrates a single cord of yarn having a geometry thatis helically organized about a central axis and composed of two levelsof twisting hierarchy. When six cords are used in parallel (e.g., Matrix1), the yarn has mechanical properties similar to a native ligament.

[0047]FIG. 2D illustrates a single cord of yarn having a geometry thatis helically organized about a central axis and composed of three levelsof twisting hierarchy. When six cords are used in parallel (e.g., Matrix2), the matrix has mechanical properties similar to a native ligament.

[0048]FIG. 3A illustrates load-elongation curves for five samples (n=5)of Matrix 1 formed from six parallel silk fibroin cords illustrated inFIG. 2A.

[0049]FIG. 3B is a chart of cycles to failure at UTS, 1680N, and 1200Nloads (n=5 for each load) illustrating Matrix 1 fatigue data. Regressionanalysis of Matrix 1 fatigue data, when extrapolated to physiologicalload levels (400 N) to predict number of cycles to failure in vivo,indicates a matrix life of 3.3 million cycles.

[0050]FIG. 3C illustrates load-elongation curves for three samples (n=3)of Matrix 2 (n=3) formed from six parallel silk fibroin cords asillustrated in FIG. 2B.

[0051]FIG. 3D is a chart of cycles to failure at UTS,2280N, 2100N and1800N loads (n=3 for each load) illustrating Matrix 2 fatigue data.Regression analysis of Matrix 2 fatigue data, when extrapolated tophysiological load levels (400 N) to predict number of cycles to failurein vivo, indicates a matrix life of greater than 10 million cycles.

[0052]FIG. 4A shows images of multiple yarn and fabric forms generatedin our laboratories. Several different yarn structures, includingvarious types of braids (i, ii, iv), a flat braid (iii), a varyingdiameter or taper braid (v), a larger (˜250 fibers) cabled two-levelbundle (vi), a parallel plied and bonded (swaged) yarn consisting24-12-fiber textured yarns (vii), a variety of twisted yarns (viii-xi),and a parallel plied and bonded (swaged) yarn consisting 24-12-fiber twolevel cabled yarns (xii).

[0053]FIG. 4B is a chart of load-elongation curves for (I) a braid (48fibers, a 4 carrier braider using twisted extracted 12 fiber yarn) andtextured yarns (48 fibers total) and (II) twisted compared to cabledyarns, 12 fibers in total-all samples were 3 cm in length.

[0054]FIG. 4C is a chart of fatigue data for small yarns, 3 cm inlength, as compared to 3B and 3D for (I) a small cable of 36 fibers and(II) a small textured yarn of 60 fibers).

[0055]FIG. 5A provides strength and stiffness data for a 36 fiber yarnas a function of 6 different strain rates at which they were tested (N=5per group).

[0056]FIG. 5B shows load-elongation curves for a 36-fiber yarn, 3 cmlong, tested at 2 of the 6 different strain rates. The data representsthe effect of the testing procedures (here, specifically strain rate) onthe reported mechanical properties (e.g. UTS) of the yarn structure.

[0057]FIG. 6A is a chart of UTS as a function of twists per inch (TPI);trend lines were generated to extrapolate data to a 4^(th) orderpolynomial—TPIs from 0-15 are shown. A maximum was observed indicatingan ordered structure where individual filaments are working in unison.

[0058]FIG. 6B is a chart of stiffness (for a 3 cm length sample) as afunction of twists per inch (TPI); trend lines were generated toextrapolate data to a 5^(th) order polynomial—TPIs from 0-15 are shown.A maximum was observed indicating that TPI could be used as a tool todesign for a specific UTS or stiffness.

[0059]FIG. 7A illustrates SEM of extracted silk fibroin prior to seedingwith cells.

[0060]FIG. 7B illustrates SEM of bone marrow stromal cells seeded andattached on silk fibroin immediately post seeding.

[0061]FIG. 7C illustrates SEM of bone marrow cells attached and spreadon silk fibroin 1 day post seeding.

[0062]FIG. 7D illustrates SEM of bone marrow stromal cells seeded onsilk fibroin 14 days post seeding forming an intact cell-extracellularmatrix sheet.

[0063]FIG. 8A illustrates a 3 cm length of the silk fibroin cordillustrated in FIG. 2C and seeded with bone marrow stromal cells,cultured for 14 days in a static environment and stained with MTT toshow even cell coverage of the matrix following the growth period.

[0064]FIG. 8B illustrates a control strand of silk fibroin cord 3 cm inlength stained with MTT.

[0065]FIG. 9A is a chart illustrating bone marrow stromal cellproliferation on silk fibroin Matrix 1 determined by total cellular DNAover 21 day culture period indicating a significant increase in cellproliferation after 21 days of culture.

[0066]FIG. 9B is a bar graph illustrating bone marrow stromal cellproliferation on silk fibroin Matrix 2 determined by total cellular DNAover 14 day culture period indicating a significant increase in cellproliferation after 14 days of culture.

[0067]FIG. 10 illustrates the ultimate tensile strength of a 30 silkfiber extracted construct that is either seeded with bone marrow stromalcells or non-seeded over 21 days of culture in physiological growthconditions.

[0068]FIG. 11A is a chart of UTS as a function of in vitro enzymaticdegradation; no strength loss was observed in the negative control, PBS.Silk lost 50% of its strength after 21 days in culture. A 1 mg/mlsolution of Protease XIV from Sigma was used.

[0069]FIG. 11B is a chart of mass loss as a function of in vitroenzymatic degradation; no strength loss was observed in the negativecontrol, PBS. 50% mass loss was observed after 41 days in culture.

[0070]FIG. 12 is a chart of UTS loss as function of in vivo degradationfollowing RGD-modified matrix implantation into a non-loadedsubcutaneous rat model for 10, 20 and 30 days. 50% strength loss wasobserved after 10 days in vivo in a non-loaded environment.

[0071]FIG. 13A shows histological sections of 12(0)×3(8) non-modifiedand RGD-modified sericin-free silk fibroin matrices after 30 days ofsubcutaneous implantation in a Lewis rat. Row I is H&E staining at 40×,row II is H&E staining at 128×, row III is collagen trichrome stainingat 128×, row IV is collagen backed out of the row III images to allowfor collagen ingrowth quantification and row V are the pixels associatedwith the cross-sections of remaining silk fibroins backed out to allowfor quantification of degradation. Upon qualitative assessment, in thesubcutaneous environment, both the non-treated and modified groupssupported cell ingrowth and collagen deposition within the matrix itselfwith limited peripheral encapsulation.

[0072]FIG. 13B quantitatively represents a 36% decrease in RGD-modifiedsilk cross-sectional area after 30 days of subcutaneous implantationindicating a significant improvement in the ability of the host todegrade the surface modified silk fibroin matrices compared tonon-treated controls.

[0073]FIG. 13C quantitatively shows a significant 63% increase incollagen deposition within the RGD-modified fibroin matrices as comparedto the non-treated controls again demonstrating the ability of themodified silk matrix to support host cell and tissue ingrowth.

[0074]FIG. 13D shows H&E staining of an extracted 36 fiber fibroin yarnimplanted intra-muscularly in the abdominal Was of a Lewis rat. Imagesare shown at 40× and 128× for both non-modified and RGD-modifiedmatrices. Results show, qualitatively, that RGD-modificationdramatically increased cell and tissue infiltration within 30 days invivo. Unlike black braided silk suture or virgin silk suture, noperipheral encapsulation or plasma cells were observed. Compared to thesubcutaneous implants, little to no cell infiltration and collagendeposition was observed in the non-treated controls indicating theeffect of implantation site in addition to surface modification.

[0075]FIG. 13E is a numerical representation of mass loss in vivo fromthe two different modification groups compared to non-treated controls.RGD modification, followed by gas plasma modification significantly(p<0.05) increased the extent of degradation after 90 days ofintramuscular implantation. However, it appears degradation was moreaggressive in the subcutaneous environment as compared to theintra-muscular environment, as was expected.

[0076]FIG. 14 illustrates gel eletrophoretic analysis of RT-PCRamplification of selected markers over time. The gel shows upregulationin both collagen types I and III expression levels normalized to thehousekeeping gene, GAPDH by bone marrow stromal cell grown on Matrix 2over 14 days in culture. Collagen type II.(as a marker for cartilage)and bone sialoprotein (as a marker of bone tissue formation) were notdetected indicating a ligament specific differentiation response by theBMSCs when cultured with Matrix 2.

[0077]FIGS. 15A and FIG. 15B illustrates a single cord of Matrix 1 (notseeded at the time of implantation) following six weeks of implantationin vivo and used to reconstruct the medial collateral ligament (MCL) ina rabbit model. FIG. 15A shows Matrix 1 fibroin fibers surrounded byprogenitor host cells and tissue ingrowth into the matrix and around theindividual fibroin fibers visualized by hematoxylin and eosin staining.FIG. 15B shows collagenous tissue ingrowth into the matrix and aroundthe individual fibroin fibers visualized by trichrome staining.

[0078]FIGS. 16A, 16B and 16C illustrate bone marrow stromal cells seededand grown on collagen fibers for 1 day (FIG. 16A) and 21 days (FIG.16B); RT-PCR (FIG. 16C) and gel electrophoretic analysis of collagen Iand III expression vs. the housekeeping gene GAPDH: a=Collagen I, day14; b=Collagen I, day 18; c=Collagen III, day 14; d=Collagen III, day18; e=GAPDH, day 14; f=GAPDH, day 18. Collagen type II (as a marker forcartilage) and bone sialoprotein (as a marker of bone tissue formation)were not detected indicating a ligament specific differentiationresponse.

[0079]FIG. 17 illustrates real-time quantitative RT-PCR at 14 days thatyielded a transcript ratio of collagen I to collagen III, normalized toGAPDH, of 8.9:1.

[0080]FIG. 18A and FIG. 18B are H&E stained cross-sections of of 6bundles of (A) 2-0 black braided silk suture and (B) RGD-surfacedmodified silk (36 fibers/bundle), respectively, 30 days followingintra-muscular implantation. 18C is RGD-modified silk pre-seeded withBMSCs for 4 weeks prior to implantation. FIG. 18A shows a typical andextensive foreign body reaction to commercially available (Ethicon,Inc.) black braided silk suture where no ingrowth or cell infiltrationcan be observed. FIG. 18B demonstrates the engineered silk's ability topromote cell and tissue ingrowth. FIGS. 18A, 18B and 18C illustratetissue response to silk fiber constructs that are coated in wax (FIG.18A), stripped of sericin and coated with RGD (FIG. 18B), and strippedof sericin and seeded with progenitor adult stem cells (FIG. 18C).

[0081] FIGS. 19A-D shows H&E stained cross sectional images at 40× (toprow, FIG. 19A & FIG. 19B) and 128× (bottom row, FIGS. 19C and 19D) oftwo yarns (4×3×3 and 12×3), each containing the same number of fibers,but organized differently with specific hierarchies followingimplantation in a rat model for 30 days. Results indication that yarndesign and structure can influence the extent of cell and tissueingrowth as the 12×3 yarn construct allowed for ingrowth, while itappears the 4×3×3 thwarted it.

[0082]FIGS. 20A, B and C are pictures of (A) single fiber wet laidnon-woven fabric extracted post fabric formation (fibers can first beextracted and formed into the non-woven—data not shown), (B) a knitfabric produced from a form of chain stitching using 12-fiber yarnextracted post fabric formation, and (C) a woven fabric produced frompre-extracted 12-fiber yarn with a 36-fiber pre-extracted yarn runningin the weft direction.

[0083]FIG. 21 is a schematic flow chart of the various methods andsequences that can be employed to create a biocompatible andbiodegradable silk fibroin matrix. For example, extract single fiber,twist into yarns and knit into fabrics OR ply yarns, twist plied yarns,form fabric and then extract. An almost infinite number of combinationexists, but all will be dependent on the hierarchy of the yarn, thenumber of fibers per level and the TPI per level as shown in Tables 4,6, 7, and 8.

DETAILED DESCRIPTION

[0084] In methods described in greater detail, below, silk fibroinfibers are aligned in a parallel orientation; the fibers can remain in astrictly parallel orientation, or they can be twisted or otherwiseintertwined to form a yarn. The yarn can include any number ofhierarchies, beginning at fiber level and expanding through bundle,strand, cord, etc., levels. Intertwining can be provided at each level.Furthermore, sericin is extracted from the silk fibers at any point inthe hierarchy up to the point where the number of fibers exceeds that atwhich the extracting solution can penetrate throughout the yarn. Themaximum number of silkworm fibroin fibers (20/22 denier as purchased)that can be combined and successfully extracted is about 50 (Table 4).These yarns can then be used as a fiber construct for, e.g., ligament ortissue reconstruction, or can be incorporated into a fabric for use,e.g., in the generation of soft tissue mesh for repairs such as herniarepair, abdominal floor reconstruction and bladder slings. Formation offiber constructs will be discussed in the context of exemplaryapplications, below.

[0085] Although much of the discussion that follows is directed to asilk-fiber-based matrix (i.e. construct, scaffold) for producing ananterior cruciate ligament (ACL), a variety of other tissues, such asother ligaments and tendons, cartilage, muscle, bone, skin and bloodvessels, can be formed using a novel silk-fiber based matrix. In thecase of the ACL, a large yarn (540-3900 fibers per yarn, before plyingin parallel; see Table 8 & 11) with multiple hierarchical levels ofintertwining and relevant physiological properties was described. Inaddition to a silk-fiber-based ACL matrix, multiple smaller yarnconfigurations (1-50 silk fibers) (Table 1, 4 & 5) with relevantphysiological properties after combining either in parallel or into aspecific fabric formation, can serve as tissue matrices for guidedtissue formation (FIG. 2A-B). In addition to silk matrices for guidedtissue formation or engineering, this work is specifically directly toproducing a variety of silk-fiber based matrices tissue supportstructures for guided tissue repair (e.g., hernia repair, bladder slingsfor urinary stress incontinence) (FIG. 2A-B & FIG. 20A-C).

[0086] Constructs (i.e. fabrics or yarns) can be surface modified orseeded with the appropriate cells (FIG. 7A-D, FIG. 8A-B & FIG. 16A-C)and exposed to the appropriate mechanical stimulation, if necessary, forproliferating and differentiating into the desired ligament, tendon orother tissue in accordance with the above-described techniques.

[0087] Additionally, the present invention is not limited to using bonemarrow stromal cells for seeding on the fiber construct, and otherprogenitor, pluripotent and stem cells, such as those in bone, muscleand skin for example, may also be used to differentiate into ligamentsand other tissues.

[0088] Fabrics can also be formed from similar constructs of purifiedfilaments, and used in various applications. Fabrics can be divided intovarious classes, including woven, non-woven, knitted fabrics, andstitch-bonded fabrics, each with numerous subtypes. Each of these typesmay be useful as an implant in particular circumstances. In discussingthese silk-based fabrics, we describe the natural silk, e.g., of Bombyxmori, as a “fibroin fiber.” The fibers should be at least one meterlong, and this length should be maintained throughout the process tofacilitate their handling during processing and incorporation into afabric. Given that a yarn may be defined as an assembly of fiberstwisted or otherwise held together in a continuous strand and that asingle fibroin fiber, as defined above, is comprised of multiple pliedbroins, sometimes from multiple cocoons, a single fibrion fiber may betermed a “yarn.” As well, fibroin fibers are twisted together orotherwise intertwined to form a “yarn.” Yarns are used to weave or knitfabrics for use in the invention. In an alternative procedure, silkyarns are disaggregated into shorter (5 mm to 100 mm) lengths or intosilk fibroin filaments. These filaments may then be (wet) laid to form anon-woven fabric (FIG. 20A).

[0089] When the yarns are formed into a fabric, the tension (force)exerted on the yarns (typically, via machinery) is no greater than theyarn's yield point (FIG. 3A-D). Accordingly, the yarns are handled atlower speeds and under smaller loads than are yarns that are typicallyused in, e.g., textile manufacturing when forming the fabric so as topreserve the integrity of the exposed fragile fibroin fibers. Likewise,contact points between handling machinery and the yarn are designed toavoid sharp angles and high-friction interactions so as to preventlousing and fraying of fibers around the perimeter of the yarn (FIG.4A-C).

[0090] Numerous applications of fabrics as implants are known in themedical and surgical arts. One example is as a support in hernia repair.For such repair, a fabric, most typically a warp-knit with a desiredstitch (e.g., an atlas stitch designed to prevent unraveling of the meshduring cutting), is sewn (or sometimes stapled or glued) or simply laidin place without tensioning, onto the inside of the abdominal wall afterit is repaired with conventional sutures. One function of the warp knitfabric is to provide short-term support for the repair. In a preferredembodiment of the present invention, the fibroin fibers within thefabric promote ingrowth of cells and subsequent tissue growth intofabric itself (FIGS. 13A & 13D) as well as through the fabric'sinterstices formed during knitting and into the region in need ofrepair. This embodiment aims to permanently strengthen the injured areathrough functional tissue ingrowth and remodeling as the silk matrixdegrades (FIGS. 13A, B & C).

[0091] Repair-strengthening fabrics are used in similar situations forrepair or support of any part of the abdominal wall, particularly inhernia repair and abdominal floor reconstruction, or in repair orsupport of other walls and septa in the body, for example of the chest,or of organs such as the heart or the bladder, particularly aftersurgery or tumor removal. Implantable fabrics can also be used tosupport bladders or other internal organs (included but not limited tothe intestines, the ureters or urethra, and the uterus) to retain themin their normal positions after surgery, damage or natural wear as aresult of age or pregnancy, or to position them in an appropriatelocation. “Organ” here includes both “solid” organs, such as a liver,and tubular organs such as an intestine or a ureter. Fabrics, especiallybulky fabrics such as some non-woven types or those that can be createdthrough 3-dimensional knitting or braiding (FIG. 4A-C), can be used tofill cavities left by surgery to provide a fiber construct onto whichcells can migrate or to which cells can be pre-attached (e.g. to improvethe rate of repair). Usage sites include cavities in both soft tissuesand hard tissues such as bone. In other cases, fabrics are used toprevent adhesions, or to prevent the attachment and/or ingrowth ofcells; this may be achieve through surface modification of the silkfibroin matrix or through the attachment of a drug or factor to thematrix.

[0092] The silk-fibroin-based fabrics of the invention can easily bemodified in several ways to enhance healing or repair at the site. Thesemodifications may be used singly or in combination. Thesilk-fibroin-based fabrics of the invention can be surface modified tosupport cell attachment and spreading, cell and tissue ingrowth andremodeling, and device biodegradation through the use of RGD peptidecoupling or gas plasma irradiation (FIGS. 13A-E). The fabrics can bemodified to carry cell-attachment factors, such as the well-knownpeptide “RGD” (arginine-glycine-aspartic acid) or-any of the manynatural and synthetic attachment-promoting materials, such as serum,serum factors and proteins including fibronectin, blood, marrow, groups,determinants, etc., known in the literature. Such materials can be inany of the usual biochemical classes of such materials, includingwithout limitation proteins, peptides, carbohydrates, polysaccharides,proteoglycans, nucleic acids, lipids, small (less than about 2000Daltons) organic molecules and combinations of these. Such plasmamodification can improve the fabric's surface functionality and/orcharge without affecting the materials bulk mechanical properties.Fabrics can be gas plasma irradiated after sericin extraction withoutcompromising the integrity of the sericin-extracted silk fibroin fibers(Table 9).

[0093] Additionally, the fabric can be treated so that it delivers adrug. Attachment of the drug to the fabric can be covalent, or covalentvia degradable bonds, or by any sort of binding (e.g., chargeattraction) or absorption. Any drug can be potentially used;non-limiting examples of drugs include antibiotics, growth factors suchas bone morphogenic proteins (BMPs) or growth differentiation factors(GDFs), growth inhibitors, chemo-attractants, and nucleic acids fortransformation, with or without encapsulating materials.

[0094] In another modification, cells can be added to the fabric beforeits implantation (FIG. 7A-D, FIG. 8A-B, and FIG. 9A-B). Cells can beseeded/absorbed on or into the fabric. Cells can also or in addition becultivated on the fabric, as a first step towards tissue replacement orenhancement. The cells may be of any type, but allogenous cells,preferably of the “immune protected,” immune privileged,” or stem celltypes are preferred, and autologous cells are particularly preferred.The cells are selected to be able to proliferate into required celltypes on or in the fiber construct (FIG. 9A-B).

[0095] Another class of modification is incorporation of other polymers(e.g. in fiber or gel form) into the fabric, to provide specificstructural properties or to modify the native surfaces of the silkfibroin and its biological characteristics (see FIG. 16A-C: seeding ofcollagen fibers with BMSCs). In one type of incorporation, fibers oryarns of silk and of another material are blended in the process ofmaking the fabric. In another type, the silk-based fibers, yarns orfabrics are coated or over-wrapped with a solution or with fibers ofanother polymer. Blending may be performed (i) randomly, for example byplying (1 or multiple fibers of) both silk and the polymer together inparallel before twisting or (ii) in an organized fashion such as inbraiding where fibers or yarns being input into the larger yarn orfabric can alternate machine feed positions creating a predicableoutcome. Coating or wrapping may be performed by braiding or cablingover a central core, where the core can be the polymer, the silk fibroinor a composit of both, depending on the desired effect. Alternatively,one yarn can be wrapped in a controlled fashion over the other polymer,where the wrapping yarn can be used to stabilize the structure. Anybiocompatible polymer is potentially usable. Examples of suitablepolymers include proteins, particularly structural proteins such ascollagen and fibrin, and strength-providing degradable syntheticpolymers, such as polymers comprising anhydrides, hydroxy acids, and/orcarbonates. Coatings may be provided as gels, particularly degradablegels, formed of natural polymers or of degradable synthetic polymers.Gels comprising fibrin, collagen, and/or basement membrane proteins canbe used. The gels can be used to deliver cells or nutrients, or toshield the surface from cell attachment. Further, proteins or peptidescan be covalently attached to the fibers or the fibers can be plasmamodified in a charged gas (e.g., nitrogen) to deposit amine groups; eachof these coatings supports cell attachment and ingrowth, as silk isnormally hydrophobic, and these coatings make the fibers morehydrophilic.

[0096] Non-limiting examples of some of these embodiments are describedin examples, below.

[0097] Wet laydown was selected for a prototype of fabric formationbecause it is the simplest procedure. The non-woven product (FIG. 20A)was created from a single silk fibroin fiber prior to extraction at thefabric level. The product is correspondingly a relatively inexpensivematerial, and can be used in applications where its low tensile strengthwould be satisfactory. When more tensile strength is needed, a non-wovenmaterial could be bonded together, as is well known for fabrics andpaper or mineralized for bone repair. Alternatively, silk yarn materialproduced by extraction of the sericin can be formed into a variety ofmore complex yarns, as described above. The size and design of the yarncan be used to control porosity, independent of non-woven machinecapabilities. The yarns can also be knit (FIG. 20B) or woven (FIG. 20C)into a fabric. One type of fabric of interest is a simple mesh, similarto gauze, which can be used by itself (e.g. as a hemostat), or todeliver cells or drugs (e.g. a clotting factor) to a site, in asituation where flexibility is important.

[0098] When strength is important, a warp knit fabric (FIG. 20B),including the familiar tricots and jerseys, having an elasticity thatcan be controlled through the helical design of the yarn used in thefabric, and typically substantial tensile strength, can be very usefulfor applications (e.g., hernia repair, bladder slings, pelvic floorreconstructions, etc.) requiring provision of mechanical support for asignificant length of time, such as months.

[0099] In other applications, the material should have little elasticityand great strength. For such fabrics, a dense weave of thick yarns isappropriate, producing a material similar to standard woven fabrics(FIG. 20C). Such a material can optionally be supplemented by, a coatingtreatment or a heat treatment to bond the crossovers of the yarnsegments, thereby preventing both raveling and stretching. Heattreatment must not entirely denature the silk protein. The fabric canoptionally be sewn, glued or stapled into place, as is currently donewith polypropylene mesh. The implant, like any of the other typesdiscussed, can be coated with various materials to enhance the localhealing and tissue ingrowth process, and/or with a coating to preventadhesion of the repair site to the viscera.

[0100] In another alternative, the fabric, mesh, non-woven, knit orother repair material can be made of unextracted silk, and then thefinished fabric can be extracted as described herein (FIG. 21 )(forexample, with alkaline soap solution at elevated temperature) to removethe immunomodulatory sericins from the material. As a furtheralternative, the extraction of the sericin can take place at anintermediate stage, such as extraction of the formed yarn, bundle, orstrand, in so far as the number of fibers does not exceed that at whichthe extracting solution can penetrate throughout the fibers (see FIG. 21for non-limiting options).

[0101] The above discussion has described making fabrics composed ofyarns, where the most-typical form of yarn in the fabric formationsdiscussed about would derive from twisting silkworm fibroin fiberstogether in an organized manner and extracting sericin. Many yarngeometries and methods of yarn formation may also be used as described(Tables 4, 5, 6, 7 & 8). Such methods may include the formation ofnon-twisted bundles of fibroin fibers, bound together by wrapping thebundles with silk or another material as discussed above. Any of theseyarns could, as described above, be formed by blending silk fibers withother materials. Further still, the fibers can be intertwined, e.g.,cabled, twisted, braided, meshed, knitted, etc. (see FIGS. 2A&B and 21).The term, “intertwined,” is used herein to indicate an organized (i.e.,non-random) repeating structure in terms of how the fibers contact andbind one another.

[0102] Blending could also be done at higher levels of organization,such as the use of filaments of different materials to form a thickeryarn, or using yarns of differing materials in weaving or knitting. Ineach case, the final material would include purified, essentiallysericin-free silk as a significant component, used for one or all of itsstrength and biocompatibility and (e.g., long-term) degradationcharacteristics (FIG. 11A-B). The other polymer or polymers are selectedfor their biocompatibility, support (or inhibition through rapid tissueformation at desired-locals) of cell attachment or infiltration (FIG.16A-C), degradation profile in vivo, and mechanical properties.Biodegradable polymers include any of the known biodegradable polymers,including natural products such as proteins, polysaccharides,glycosaminoglycans, and derivatized natural polymers, e.g., celluloses;and biodegradable synthetic polymers and copolymers includingpolyhydroxy acids, polycarbonates, polyanhydrides, some polyamides, andcopolymers and blends thereof. In particular, collagen and elastin aresuitable proteins.

[0103] Silk-containing fabric constructs/matrices used for tissue repairmay be treated so that they contain cells at the time of implantation(FIG. 7A-D, FIG. 8A-B, FIG. 9A-B, & FIG. 18C) to improve tissue outcomesin vivo. The cells may be xenogenic, more preferably allogenic, and mostpreferably autologous. Any type of cell is potentially of use, dependingon the location and the intended function of the implant. Pluripotentcells are preferred when the appropriate differentiation cues arepresent or provided in the environment. Other cell types includeosteogenic cells, fibroblasts, and cells of the tissue type of theimplantation site.

[0104] While silk from Bombyx mori and other conventional silkworms hasbeen described, any source of silk or silk-derived proteins can be usedin the invention, as long as it provokes no more than a mild foreignbody reaction on implantation (i.e., is biocompatible)(see FIGS. 18B &C). These include without limitation silks from silkworms, spiders, andcultured cells, particularly genetically engineered cells, andtransgenic plants and animals. Silk produced by cloning may be from fullor partial sequences of native silk-line genes, or from synthetic genesencoding silk-like sequences.

[0105] While in many cases only a single fabric type will be used information of a medical device or prosthesis, it may be useful in somecases to use two or more types of fabric in a single device. Forexample, in hernia repair, it is desirable to have the tissue-facingside of the repair fabric attract cells, while the peritoneal faceshould repel cells, to prevent adhesions. This effect can be achieved byhaving one layer of silk that does not attract cells, and another layerthat does (for example, an untreated layer and an RGD-containing layer,as in the example, below). Another example includes formation of abladder sling. The basic sling should be conforming and somewhatelastic, and have a long projected lifetime. However, the face of thesling closest to the bladder should have as little texture as feasible.This can be accomplished by placing a layer of thin but tightly woven,non-woven or knitted fabric, fabricated from a yarn having a smalldiameter (e.g., a single fiber), of the invention in the sling where itwill contact the bladder. The non-woven fabric should be of as small agauge (denier) as feasible. Numerous other situations needing two ormore types of fabric are possible.

[0106] Examples of the above-described structures were fabricated andevaluated in a series of tests. In a first example, a fabric was formedfrom purified silk fibrils. First, raw silk was processed into purifiedfibroin fibrils. Raw silkworm fibers were extracted in an aqueoussolution of 0.02 M Na2CO3 and 0.3% w/v IVORY soap solution for 60minutes at 90 degrees. C. The extracted fibers were rinsed with water tocomplete the extraction of the glue-like sericin protein. The resultingsuspension of fibrils was wet-laid on a screen, needle-punched, anddried (FIG. 20A). The resulting fleecy material felt somewhat like woolto the touch, and was very porous. It was sufficiently interbonded byentanglement and needling that it was easily handled and cut to adesired shape.

[0107] In another example, the purified silk fibroin fibrils weretreated with cell attracting agents (Table 9). First, yarns were made bytwisting purified fibers of silk fibroin together. Some yarns were madeof filaments that were derivatized with the peptide RGD to attractcells, using procedures described in Sofia et al, J. Biomed. Mater. Res.54: 139-148, 2001. Sections of treated and untreated (black braided silksuture) yarns were implanted in the abdominal wall of rats (FIG. 18A-C).After 30 days of implantation, the black braided sutures containedcompact fibril bundels, with cell infiltration between fibril bundlesbut not within them. In contrast, the RGD treated fibril bundles wereextensively invaded by host cells, and were expanded and non-compact(FIG. 13A-E, 18B), but were not yet significantly degraded (FIG. 13A-E).

[0108] This example illustrates the use of derivatization to control therate of degradation of implanted silk fibroin fibrils, as well asdemonstrating the ability of derivatized fibrils to recruit cells to afabric-like structure. Clearly, greater specificity of recruitment canbe obtained by using a more specific attractant. Similar techniques(chemical derivatization) or simpler methods such as absorption,adsorption, coating, and imbibement, can be used to provide othermaterials to the implantation site.

[0109] Each of the samples reported in the Tables below, was prepared inaccordance with the above description, wherein sericin was removed over60 minutes at a temperature of 90°±2° C. Using a temperature in thisrange for a sufficient period of time has been found to produce fibersfrom which sericin is substantially removed (FIG. 1A-C, Table 1, 2,3)(to produce a fiber construct that is substantially free of sericin soas not to produce a significant immunological response and not tosignificantly impede the biodegradeability of the fiber) whilesubstantially preserving the mechanical integrity of the fibroin (Table1). Note that when temperatures reach 94° C. (Table 1), UTS was notdramatically affected; however, stiffness significantly declinedindicating a silk thermo sensitive at temperatures of 94° C. and above.The fibers in each group were manually straightened (i.e., madeparallel) by pulling the ends of the fibers; alternatively,straightening could easily have been performed via an automated process.The force applied was marginally greater than what was required tostraighten the group.

[0110] The sample geometry designations in all Tables reflect thefollowing constructs: # of fibers (tpi at fiber level in S direction)×#of groups (tpi at group level in Z direction)×# of bundles (tpi atbundle level in S direction)×# of strand (tpi at stand level in Zdirection)× etc., wherein the samples are twisted between levels unlessotherwise indicated. The twist-per-inch designation, such as 10 s×9 ztpi, reflects (the number of twists of the fibers/inch within thegroup)×(the number of twists of the groups/inch within the bundle). Ineach sample, the pitch of the twist is substantially higher than isordinarily found in conventional yarns that are twisted at a low pitchintended merely to hold the fibers together. Increasing the pitch of thetwists (i.e., increasing the twists per inch) decreases the tensilestrength, but also further decreases the stiffness and increases theelongation at break of the construct.

[0111] The ultimate tensile strength (UTS), percent elongation at break(% Elong), and stiffness were all measured using an INSTRON 8511servohydraulic material testing machine with FAST-TRACK software, whichstrained the sample at the high rate of ˜100% sample length per secondin a pull-to-failure analysis. In other words, up to the point offailure, the sample is stretched to double its length every second,which greatly restricts the capacity of the sample to relax and reboundbefore failure. However, FIG. 5A-B demonstrates the effect of strainrate can have on observed mechanical properties as well as wet or drytesting conditions which were shown (FIG. 6A-B) to have a dramaticeffect on silk matrix UTS and stiffness. Consistency is needed ifcomparisons are to be made between data sets. The resulting data wasanalyzed using Instron Series IX software. Ultimate tensile strength isthe peak stress of the resulting stress/strain curve, and stiffness isthe slope of the stress/strain plot up to the yield point. Unlessspecified, at least an N=5 was used for all tested groups to generateaverages and standard deviations. Standard statistical methods wereemployed to determine if statistically significant differences existedbetween groups, e.g., Student's t-test, one-way ANOVA.

[0112] The fibroin fibers in the samples in all of the above Tables andFigures (and throughout this disclosure) are native (i.e., the fibersare not dissolved and reformed); dissolution and reformulation of thefibers results in a different fiber structure with different mechanicalproperties after reforming. Surprisingly, these samples demonstrate thatyarns of silk fibroin fibers, from which sericin has been completely ornearly completely removed, can possess high strengths and othermechanical properties that render the yarns suitable for variousbiomedical applications (Table 4, FIG. 2A-D & FIG. 20A-C), such as forforming a fiber construct or support for ligament replacement, herniarepair or pelvic floor reconstruction. Previously, it was believed thatfibroin needed to be dissolved and extruded into a reformulated fiber toprovide desired mechanical properties. Fatigue strength has generallybeen found to suffer in such reformed fibroin fibers. The methods of thepresent invention, allow for sericin removal without a significant lossof strength (Tables 1 & 4; FIGS. 3A-D & 4A-B).

[0113] In Table 8, samples 1 and 2 compare the properties of a 3-fibergroup (sample 1) with those of a 4-fiber group (sample 2). Sample 2 hada square configuration of fibers, while the fibers of-sample 1 had atriangular configuration. As shown in the Table, the addition of theextra fiber in sample 2 lowered the per-fiber stiffness of the sampledemonstrating the ability to control yarn and fabric properties throughhierarchical design.

[0114] Table 4 illustrates the effects of different configurations ofcabled-fiber constructs and a twisted-fiber geometry. Note, inparticular, samples 7 and 8 include the same number of fibers and thesame number of geometrical levels. The twisted-fiber geometry of sample8 offers greater UTS and greater stiffness, while the cabled geometry ofsample 7 has lower strength and lower stiffness. Of samples 7-9, thecabled geometry of sample 7 has the highest strength-to-stiffness ratio;for use as an ACL fiber construct, a high strength-to-stiffness ratio isdesired (i.e., possessing a high strength and low stiffness).

[0115] Tables 1 and 4 demonstrate the effect of sericin extraction onthe fibers. All samples were immersed in an extraction solution, asdescribed in Table 1. Samples 1-5 were immersed in a bath at roomtemperature, at 33° C. and 37° C. These temperatures are believed to betoo low to provide significant sericin extraction. Samples 6-9 wereextracted at 90° C., where complete sericin extraction is believed to beattainable, but for varying times. Similarly sample 10 was extracted-atthe slightly higher temperature of 94° C. The data suggests that 30 to60 min at 90° C. is sufficient to significantly remove sericin (seeTables 2&3) and that 94° C. may be damaging the protein structure ofsilk as shown by a dramatic decrease in stiffness.

[0116] Finally, samples 11 to 16 have comparable cabled geometries; thefibers of samples 12, 14, and 16 were extracted, whereas the fibers ofsamples 11, 13, and 15 were not. As can be seen in the Table, theextraction appears to have had little effect on (high) ultimate tensilestrengths per fiber.

[0117] The fibers of sample 10 of Table 4 were subject to acurl-shrinking procedure, wherein the fibers were twisted in onedirection and then in the opposite direction, rapidly; the fibers wherethen heated to lock in the twist structure and tested non-extracted. Thestrength and stiffness of the resulting yarn were comparatively lowerthan most of the other non-extracted yarns tested. However, Tables 6&7show the fibroins remarkable ability, post extraction, to withstand upto 30 TPI. Table 6 shows the ordering effect TRI has on silk matriceslikely due to the ordering of the multifilament structure followingextraction.

[0118]FIG. 10 demonstrates the properties of a group of 30 parallelfibroin fibers seeded and non-seeded in culture conditions for 21 days.These three samples exhibited very similar mechanical properties,thereby reflecting little if any degradation of silk matrices due tocell growth thereon or due to time in vitro. Stiffness values are likelymuch lower in this experiment in comparison with the other samples as aresult of the 21 day wet incubation prior to mechanical testing (seeTable 5).

[0119] Table 4, samples 14-16 are all braided samples. The fibers ofsample 14 were braided from eight carriers, with a spool mounted on eachcarrier, wherein two fibers were drawn from each spool. The fibers ofsample 15 were drawn from 16 carriers, with a spool mounted on eachcarrier; again, two fibers were drawn from each spool. Finally, sample16 was formed from 4 yarns, each yarn comprising 3 twisted groups offour fibers (providing a total of 12 fibers per yarn); each of the yarnswas drawn from a separate spool and carrier.

[0120] Table 9 demonstrates the effect of surface modification. Thedesignation, “PBS,” reflects that the samples were immersed in aphosphate-buffered saline solution for about 24 hours before testing.The effect of exposing the samples to the saline solution was measuredand provided an indication that the fiber construct can maintain itsmechanical properties and substantially preserve the inherent proteinstructure in a saline environment (e.g., inside a human body). The “RGD”designation reflects that the samples were immersed in an arg-gly-asp(RGD) saline solution for about 24 hours before testing. RGD can beapplied to the construct to attract cells to the construct and therebypromote cell growth thereon. Accordingly, any effect of RGD on themechanical properties of the construct is also of interest, though nosignificant degradation of the construct was apparent. Accordingly,these samples offer evidence that prolonged exposure to a salinesolution or gas ethylene oxide sterilization or to an RGD solutionresults in little, if any, degradation of the material properties of thefiber constructs. Though, the data associated with samples 28 and 29,wherein the geometrical hierarchy was extended to a higher level, revealthat the UTS/fiber drops as higher levels (and increased overall fibercount) are reached. This is an effect of heiarchical design (Table 8)rather than surface modification.

[0121] Table 4, samples 18 through 23 were tensioned under 6 pounds ofconstant force for 1, 2, 3, 4, 5 and 6 days, respectively, beforetesting to evaluate the effect of tension on the mechanical propertiesover time. From the data, there does not appear to be much if any changein the material properties of the construct as the pretension procedureis extended over longer periods of time. Sample 25 was also“pre-tensioned” (after twisting) at 6 pounds force for a day beforetesting; for comparison, sample 24, which had an identical geometricalconfiguration was not pre-tensioned. Samples 24 and 25 accordinglyreveal the effect of pre-tensioning the construct to remove the slack inthe structure, which results in a slight reduction in boththe-construct's UTS and its elongation at break.

[0122] The silk-fiber-based construct serves as a matrix forinfiltrating cells or already infiltrated or seeded with cells, such asprogenitor, ligament or tendon fibroblasts or muscle cells, which canproliferate and/or differentiate to form an anterior cruciate ligament(ACL) or other desired tissue type. The novel silk-fiber-based constructis designed having fibers in any of a variety of yarn geometries, suchas a cable, or in an. intertwined structure, such as twisted yarn,braid, mesh-like yarn or knit-like yarn. The yarn exhibits mechanicalproperties that are identical or nearly identical to those of a naturaltissue, such as an anterior cruciate ligament (see Table 4, 1, infra);and simple variations in fiber construct organization and geometry canresult in the formation of any desired tissue type (see Table 10,infra). Alternatively, a plurality of yarns can be formed into a fabricor other construct that is implanted to position or support an organ.Additionally, the construct can be used to fill internal cavities aftersurgery or to prevent tissue adhesions or promote the attachment oringrowth of cells.

[0123] Pluripotent bone marrow stromal cells (BMSCs) that are isolatedand cultured as described in the following example can be seeded on thesilk-fiber construct and cultured in a bioreactor under staticconditions. The cells seeded onto the fiber construct, if properlydirected, will undergo ligament and tendon specific differentiationforming viable and functional tissue. Moreover, the histomorphologicalproperties of a bioengineered tissue produced in vitro generated frompluripotent cells within a fiber construct are affected by the directapplication of mechanical force to the fiber construct during tissuegeneration. This discovery provides important new insights into therelationship between mechanical, stress, biochemical and cellimmobilization methods and cell differentiation, and has applications inproducing a wide variety of ligaments, tendons and tissues in vitro frompluripotent cells.

[0124] A fiber construct comprising silk fibers having a cable geometry,is illustrated in FIGS. 2C and 2D. The fiber construct comprises ahierarchy in terms of the way that fibers are grouped in parallel andtwisted and how the resultant group is grouped and twisted, etc., acrossa plurality of levels in the hierarchy, as is further explained, below.The silk fibers are first tensioned in parallel using, for example, arack having spring-loaded clamps that serve as anchors for the fibers.The rack can be immersed in the sericin-extraction solution so that theclamps can maintain a constant tension on the fibers through extraction,rinsing and drying.

[0125] The extraction solution can be an alkaline soap solution ordetergent and is maintained at about 90° C. The rack is immersed in thesolution for a period of time (e.g., at least 0.5 to 1 hr, depending onsolution flow and mixing conditions) that is sufficient to remove all(±0.4% remaining, by weight) or substantially all sericin (allowing forpossible trace residue) from the fibers. Following extraction, the rackis removed from the solution and the fibers are rinsed and dried.Computer-controlled twisting machines, each of which mounts the fibersor constructs of fibers about a perimeter of a disc and rotates the discabout a central axis to twist the fibers (i.e. cabling) or constructs offibers twisted about each other according to standard processes used inthe textile industry, though at a higher pitch rate for the twists(e.g., between about 0 and about 11.8 twists per cm) than is typicallyproduced in traditional yarns. The cabling or twist rate, however,should not be so high as to cause plastic deformation of the fibers as aresult of the balloon tension created as the yarn is let-off from thefeed spool prior to twisting or cabling.

[0126] Extraction can be performed at any level of the constructprovided that the solution can penetrate through the construct to removethe sericin from all fibers. It is believed that the upper limit for thenumber of fibers in a compact arrangement that can still be fullypermeated with the solution is about 20-50 fibers. Though, of course,those fibers can be arranged as one group of 20 parallel fibers or, forexample, as 4 groups of 5 parallel fibers, wherein the groups may betwisted, or even a construct comprising a still higher level such as 2bundles of 2 groups of 5 fibers, wherein the groups and bundles may betwisted. Increasing the number of hierarchical levels in the structurecan also increase the void space, thereby potentially increasing themaximum number of fibers from which sericin can be fully extracted from20 to 50 fibers.

[0127] Because the sericin, in some cases, is removed from the constructafter fibers are grouped or after a higher-level construct is formed,there is no need to apply wax or any other type of mechanicallyprotective coating on the fibers or in order to also form a barrier toprevent contact with sericin on the fibers; and the construct can befree of coatings, altogether (particularly being free of coatings thatare not fully degraded by the body or cause an inflammatory response).

[0128] As described in the examples below, mechanical properties of thesilk fibroin (as illustrated in FIGS. 1A, 1B and 1C) were characterized,and geometries for forming applicable matrices for ACL engineering werederived using a theoretical computational model (see FIG. 1D). Asix-cord construct was chosen for use as an ACL replacement to increasematrix surface area and to enhance support for tissue in-growth. Twoconstruct geometrical hierarchies for ACL repair comprise the following:

[0129] Matrix 1: 1 ACL yarn=6 parallel cords; 1 cord=3 twisted strands(3 twists/cm); 1 strand=6 twisted bundles (3 twists/cm); 1 bundle=30parallel washed fibers; and

[0130] Matrix 2: 1 ACL yarn=6 parallel cords; 1 cord=3 twisted strands(2 twists/cm); 1 strand=3 twisted bundles (2.5 twists/cm); 1 bundle=3groups (3 twists/cm); 1 group=15 parallel extracted silk fibroin fibers.

[0131] The number of fibers and geometries for Matrix 1 and Matrix 2were selected such that the silk prostheses are similar to the ACLbiomechanical properties in ultimate tensile strength, linear stiffness,yield point and % elongation at break, serving as a solid starting pointfor the development of a tissue engineered ACL. The effects ofincreasing number of fibers, number of levels, and amount of twisting oneach of these biomechanical properties are shown in Table 8 and Tables6&7, respectively.

[0132] The ability to generate two matrices with differing geometriesboth resulting in mechanical properties that mimic properties of the ACLindicates that a wide variety of geometrical configurations exist toachieve the desired mechanical properties. Alternative geometries forany desired ligament or tendon tissue may comprise any number,combination or organization of cords, strands, bundles, groups andfibers (see Table 10, infra) that result in a fiber construct withapplicable mechanical properties that mimic those of the ligament ortendon desired. For example, one(1) ACL prosthesis may have any numberof cords in parallel provided there is a mean for anchoring the finalfiber construct in vitro or in vivo. Further, various numbers oftwisting levels (where a single level is defined as a group, bundle,strand or cord) for a given geometry can be employed provided the fiberconstruct results in the desired mechanical properties. Furthermore,there is a large degree of freedom in designing the fiber constructgeometry and organization in engineering an ACL prosthesis; accordingly,the developed theoretical computational model can be used to predict thefiber construct design of a desired ligament or tendon tissue (see theexample, below). For example when multiple smaller matrix bundles aredesired (e.g., 36 fibers total) with only two levels of hierarchy topromote ingrowth; a TPI of 8-11 or -3-4 twists per cm is required andcan be predicted by the model without the need for empirical work.

[0133] Consequently, a variation in geometry (i.e., the number of cordsused to make a prosthesis or the number of fibers in a group) can beused to generate matrices applicable to most ligaments and tendons. Forexample, for smaller ligaments or tendons of the hand, the geometry andorganization used to generate a single cord of Matrix 1 (or two cords orthree cords, etc.) may be appropriate given the fiber construct'sorganization results in mechanical properties suitable for theparticular physiological environment. Specifically, to accommodate asmaller ligament or tendon compared to Matrix 1 or Matrix 2, less fibersper level would be used to generate smaller bundles or strands. Multiplebundles could then be used in parallel. In the case of a larger ligamentsuch as the ACL, it might be desirable to have more smaller bundlestwisted at higher TPIs to reduce stiffness and promote ingrowth then tohave fewer larger bundles where ingrowth cannot occur thereby limiteddegradation of the matrix.

[0134] The invention is not, however, limited with respect to the cablegeometry as described, and any geometry or combination of geometries(e.g., parallel, twisted, braided, mesh-like) can be used that resultsin fiber construct mechanical properties similar to the ACL (i.e.,greater than 2000 N ultimate tensile strength, between 100-600 N/mmlinear stiffness for a native ACL or commonly used replacement graftsuch as the patellar tendon with length between 26-30 mm) or to thedesired ligament and tendon that is to be produced. The number of fibersand-the geometry of both Matrix 1 and Matrix 2 were selected to generatemechanically appropriate ACL matrices, or other desired ligament ortendon matrices [e.g., posterior cruciate ligament (PCL)]. For example,a single cord of the six-cord Matrix 1 construct was used to reconstructthe medial collateral ligament (MC) in a rabbit (see FIG. 15A and FIG.15B). The mechanical properties of the silk six-cord constructs ofMatrix 1 and Matrix 2 are described in Table 10 and in FIGS. 3A-3D, asis further described in the example, infra. Additional geometries andtheir relating mechanical properties are listed in Table 11 as anexample of the large degree of design freedom that would result in afiber construct applicable in ACL tissue engineering in accordance withmethods described herein.

[0135] Advantageously, the silk-fiber based fiber construct can consistsolely of silk. Types and sources of silk include the following: silksfrom silkworms, such as Bombyx mori and related species; silks fromspiders, such as Nephila clavipes; silks from genetically engineeredbacteria, yeast mammalian cells, insect cells, and transgenic plants andanimals; silks obtained from cultured cells from silkworms or spiders;native silks; cloned full or partial sequences of native silks; andsilks obtained from synthetic genes encoding silk or silk-likesequences. In their raw form, the native silk fibroins obtained from theBombyx mori silkworms are coated with a glue-like protein calledsericin, which is completely or essentially completely extracted fromthe fibers before the fibers that make up the fiber construct are seededwith cells.

[0136] The fiber construct can comprise a composite of: (1) silk andcollagen fibers; (2) silk and collagen foams, meshes, or sponges; (3)silk fibroin fibers and silk foams, meshes, or sponges; (4) silk andbiodegradable polymers [e.g., cellulose, cotton, gelatin, poly lactide,poly glycolic, poly(lactide-co-glycolide), poly caproloactone,polyamides, polyanhydrides, polyaminoacids, polyortho esters, polyacetals, proteins, degradable polyurethanes, polysaccharides,polycyanoacrylates, Glycosamino glycans (e.g., chrondroitin sulfate,heparin, etc.), Polysaccharides (native, reprocessed or geneticallyengineered versions: e.g., hyaluronic acid, alginates, xanthans, pectin,chitosan, chitin, and the like), elastin (native, reprocessed orgenetically engineered and chemical versions), and collagens (native,reprocessed or genetically engineered versions], or (5) silk andnon-biodegradable polymers (e.g., polyamide, polyester, polystyrene,polypropylene, polyacrylate, polyvinyl, polycarbonate,polytetrafluorethylene, or nitrocellulose material. The compositegenerally enhances fiber construct properties such as porosity,degradability, and also enhances cell seeding, proliferation,differentiation or tissue development. FIGS. 16A, 16B and 16C illustratethe ability of collagen fibers to support BMSC growth and ligamentspecific differentiation.

[0137] The fiber construct can also be treated to enhance cellproliferation and/or tissue differentiation thereon. Exemplary fiberconstruct treatments for enhancing cell proliferation and tissuedifferentiation include, but are not limited to, metals, irradiation,crosslinking, chemical surface modifications [e.g., RGD (arg-gly-asp)peptide coating, fibronectin coating, coupling growth factors], andphysical surface modifications.

[0138] A second aspect of this disclosure relates to a mechanically andbiologically functional ACL formed from a novel silk-fiber-based fiberconstruct and autologous or allogenic (depending on the recipient of thetissue) bone marrow stromal cells (BMSCs) seeded on the fiber construct.The silk-fiber-based fiber construct induces stromal celldifferentiation towards ligament lineage without the need for anymechanical stimulation during bioreactor cultivation. BMSCs seeded onthe silk-fiber-based fiber construct and grown in a petri dish begin toattach and spread (see FIGS. 7A-D); the cells proliferate to cover thefiber construct (see FIGS. 8A-B, FIG. 9A and FIG. 9B) and differentiate,as shown by the expression of ligament specific markers (see FIG. 14).Markers for cartilage (collagen type II) and for bone (bonesialoprotein) were not expressed (see FIG. 14). Data illustrating theexpression of ligament specific markers is set forth in an example,below.

[0139] Another aspect of this disclosure relates to a method forproducing an ACL ex vivo. Cells capable of differentiating into ligamentcells are grown under conditions that simulate the movements and forcesexperienced by an ACL in vivo through the course of embryonicdevelopment into mature ligament function. Specifically, under sterileconditions, pluripotent cells are seeded within a three-dimensionalsilk-fiber-based fiber construct to which cells can adhere and which isadvantageously of cylindrical shape. The three-dimensionalsilk-fiber-based fiber construct used in the method serves as apreliminary fiber construct, which is supplemented and possibly evenreplaced by extracellular fiber construct components produced by thedifferentiating cells. Use of the novel silk-fiber-based fiber constructmay enhance or accelerate the development of the ACL. For instance, thenovel silk-fiber-based fiber construct can be designed to possessspecific mechanical properties (e.g., increased tensile strength) sothat it can withstand strong forces prior to reinforcement fromextracellular (e.g., collagen and tenascin) fiber construct components.Other advantageous properties of the novel silk-fiber based preliminaryfiber construct include, without limitation, biocompatibility andsusceptibility to biodegradation.

[0140] The pluripotent cells may be seeded within the preliminary fiberconstruct either pre- or post-fiber construct formation, depending uponthe particular fiber construct used and upon the method of fiberconstruct formation. Uniform seeding is usually preferable. In theory,the number of cells seeded does not limit the final ligament produced;however, optimal seeding may increase the rate of generation. Optimalseeding amounts will depend on the specific culture conditions. Thefiber construct can be seeded with from about 0.05 to 5 times thephysiological cell density of a native ligament.

[0141] One or more types of pluripotent cells are used in the method.Such cells have the ability to differentiate into a wide variety of celltypes in response to the proper differentiation signals and to expressligament specific markers. More specifically, the method uses cells,such as bone marrow stromal cells, that have the ability todifferentiate into cells of ligament and tendon tissue. If the resultingbioengineered ligament is to be transplanted into a patient, the cellsshould be derived from a source that is compatible with the intendedrecipient. Although the recipient will generally be a human,applications in veterinary medicine also exist. The cells can beobtained from the recipient (autologous), although compatible donorcells may also be used to make allogenic ligaments. For example, whenmaking allogenic ligaments (e.g., using cells from another human such asbone marrow stromal cells isolated from donated bone marrow or ACLfibroblasts isolated from donated ACL tissue), human anterior cruciateligament fibroblast cells isolated from intact donor ACL tissue (e.g.,cadaveric or from total knee transplantations), ruptured ACL tissue(e.g., harvested at the time of surgery from a patient undergoing ACLreconstruction) or bone marrow stromal cells may be used. Thedetermination of compatibility is within the means of the skilledpractitioner.

[0142] Ligaments or tendons including, but not limited to, the posteriorcruciate ligament, rotator cuff tendons, medial collateral ligament ofthe elbow and knee, flexor tendons of the hand, lateral ligaments of theankle and tendons and ligaments of the jaw or temporomandibular jointother than ACL, cartilage, bone and other tissues may be engineered withthe fiber construct in accordance with methods of this disclosure. Inthis manner, the cells to be seeded on the fiber construct are selectedin accordance with the tissue to be produced (e.g., pluripotent or ofthe desired tissue type). Cells seeded on the fiber construct, asdescribed herein, can be autologous or allogenic. The use of autologouscells effectively creates an allograft or autograft for implantation ina recipient.

[0143] As recited, to form an ACL, cells, such as bone marrow stromalcells, are seeded on the fiber construct. Bone marrow stromal cells area type of pluripotent cell and are also referred to in the art asmesenchymal stem cells or simply as stromal cells. As recited, thesource of these cells can be autologous or allogenic. Additionally,adult or embryonic stem or pluripotent cells can be used if the properenvironment (either in vivo or in vitro), seeded on the silk-fiber basedfiber construct, can recapitulate an ACL or any other desired ligamentor tissue in extracellular fiber construct composition (e.g., protein,glycoprotein content), organization, structure or function.

[0144] Fibroblast cells can also be seeded on the inventive fiberconstruct. Since fibroblast cells are often not referred to aspluripotent cells, fibroblasts are intended to include mature human ACLfibroblasts (autologous or allogenic) isolated from ACL tissue,fibroblasts from other ligament tissue, fibroblasts from tendon tissue,from neonatal foreskin, from umbilical cord blood, or from any cell,whether mature or pluripotent, mature dedifferentiated, or geneticallyengineered, such that when cultured in the proper environment (either invivo or in vitro), and seeded on the silk-fiber based fiber construct,can recapitulate an ACL or any other desired ligament or tissue inextracellular fiber construct composition (e.g., protein, glycoproteincontent), organization, structure or function.

[0145] The faces of the fiber construct cylinder are each attached toanchors, through which a range of forces is to be applied to the fiberconstruct. To facilitate force delivery to the fiber construct, theentire surface of each respective face of the fiber construct cancontact the face of the respective anchors. Anchors with a shape thatreflects the site of attachment (e.g., cylindrical) are best suited foruse in this method. Once assembled, the cells in the anchored fiberconstruct are cultured under conditions appropriate for cell growth andregeneration. The fiber construct is subjected to one or more mechanicalforces applied through the attached anchors (e.g., via movement of oneor both of the attached anchors) during the course of culture. Themechanical forces are applied over the period of culture to mimicconditions experienced by the native ACL or other tissues in vivo.

[0146] The anchors must be made of a material suitable for fiberconstruct attachment, and the resulting attachment should be strongenough to endure the stress of the mechanical forces applied. Inaddition, the anchors can be of a material that is suitable for theattachment of extracellular fiber construct that is produced by thedifferentiating cells. The anchors support bony tissue in-growth (eitherin vitro or in vivo) while anchoring the developing ligament. Someexamples of suitable anchor material include, without limitation,hydroxyappatite, Goinopra coral, demineralized bone, bone (allogenic orautologous). Anchor materials may also include titanium, stainlesssteel, high density polyethylene, DACRON and TEFLON.

[0147] Alternatively, anchor material may be created or further enhancedby infusing a selected material with a factor that promotes eitherligament fiber construct binding or bone fiber construct binding orboth. The term infuse is considered to: include any method ofapplication that appropriately distributes the factor onto the anchor(e.g., coating, permeating, contacting). Examples of such factorsinclude without limitation, laminin, fibronectin, any extracellularfiber construct protein that promotes adhesion, silk, factors thatcontain arginine-glycine-aspartate (RGD) peptide binding regions or theRGD peptides themselves. Growth factors or bone morphogenic protein canalso be used to enhance anchor attachment. In addition, anchors may bepre-seeded with cells (e.g., stem cells, ligament cells, osteoblasts,osteogenic progenitor cells) that adhere to the anchors and bind thefiber construct, to produce enhanced fiber construct attachment both invitro and in vivo.

[0148] An exemplary anchor system is disclosed in applicant's co-pendingapplication U.S. Ser. No. 09/950,561, which is incorporated herein byreference in its entirety. The fiber construct is attached to theanchors via contact with the anchor face or alternatively by actualpenetration of the fiber construct material through the anchor material.Because the force applied to the fiber construct via the anchorsdictates the final ligament produced, the size of the final ligamentproduced is, in part, dictated by the size of the attachment site of theanchor. An anchor of appropriate size to the desired final ligamentshould be used. An example of an anchor shape for the formation of anACL is a cylinder. However, other anchor shapes and sizes will alsofunction adequately. For example, anchors can have a size andcomposition appropriate for direct insertion into bone tunnels in thefemur and tibia of a recipient of the bioengineered ligament.

[0149] Alternatively, anchors can be used only temporarily during invitro culture, and then removed when the fiber construct alone isimplanted in vivo.

[0150] Further still, the novel silk-fiber-based fiber construct can beseeded with BMSCs and cultured in a bioreactor. Two types of growthenvironments currently exist that may be used in accordance with methodsof this disclosure: (1) the in vitro bioreactor apparatus system, and(2) the in vivo knee joint, which serves as a “bioreactor” as itprovides the physiologic environment including progenitor cells andstimuli (both chemical and physical) necessary for the development of aviable ACL given a fiber construct with proper biocompatible andmechanical properties. The bioreactor apparatus provides optimal cultureconditions for the formation of a ligament in terms of differentiationand extracellular fiber construct (ECM) production, and which thusprovides the ligament with optimal mechanical and biological propertiesprior to implantation in a recipient. Additionally, when the silk-fiberbased fiber construct is seeded and cultured with cells in vitro, apetri dish may be considered to be the bioreactor within whichconditions appropriate for cell growth and regeneration exist, i.e., astatic environment.

[0151] Cells can also be cultured on the fiber construct fiber constructwithout the application of any mechanical forces, i.e., in a staticenvironment; For example, the silk-fiber based fiber construct alone,with no in vitro applied mechanical forces or stimulation, when seededand cultured with BMSCs, induces the cells to proliferate and expressligament and tendon specific markers (see the examples, describedherein). The knee joint may serve as a physiological growth anddevelopment environment that can provide the cells and the correctenvironmental signals (chemical and physical) to the fiber constructfiber construct such that an ACL technically develops. Therefore, theknee joint (as its own form of bioreactor) plus the fiber construct(either non-seeded, seeded and not differentiated in vitro, or seededand differentiated in vitro prior to implantation) will result in thedevelopment of an ACL, or other desired tissue depending upon the celltype seeded on the fiber construct and the anatomical location of fiberconstruct implantation. FIG. 15A-B illustrates the effects of the medialcollateral knee joint environment on medial collateral ligament (MCL)development when only a non-seeded silk-based fiber construct withappropriate MCL mechanical properties is implanted for 6 weeks in vivo.Whether the cells are cultured in a static environment with nomechanical stimulation applied, or in a dynamic environment, such as ina bioreactor apparatus, conditions appropriate for cell growth andregeneration are advantageously present for the engineering of thedesired ligament or tissue.

[0152] In experiments described in the examples, below, the appliedmechanical stimulation was shown to influence the morphology, andcellular organization of the progenitor cells within the resultingtissue. The extracellular fiber construct components secreted by thecells and the organization of the extracellular fiber constructthroughout the tissue was also significantly influenced by the forcesapplied to the fiber construct during tissue generation. During in vitrotissue generation, the cells and extracellular fiber construct alignedalong the axis of load, reflecting the in vivo organization of a nativeACL that is also along the various load axes produced from natural kneejoint movement and function. These results suggest that the physicalstimuli experienced in nature by cells of developing tissue, such as theACL, play a significant role in progenitor cell differentiation andtissue formation. They further indicate that this role can beeffectively duplicated in vitro by mechanical manipulation to produce asimilar tissue. The more closely the forces produced by mechanicalmanipulation resemble the forces experienced by an ACL in vivo, the moreclosely the resultant tissue will resemble a native ACL.

[0153] When mechanical stimulation is applied in vitro to the fiberconstruct via a bioreactor, there exists independent but concurrentcontrol over both cyclic and rotation strains as applied to one anchorwith respect to the other anchor. Alternatively, the fiber constructalone may be implanted in vivo, seeded with ACL cells from the patientand exposed in vivo to mechanical signaling via the patient.

[0154] When the fiber construct is seeded with cells prior toimplantation, the cells are cultured within the fiber construct underconditions appropriate for cell growth and differentiation. During theculture process, the fiber construct may be subjected to one or moremechanical forces via movement of one or both of the attached anchors.The mechanical forces of tension, compression, torsion and shear, andcombinations thereof, are applied in the appropriate combinations,magnitudes, and frequencies to mimic the mechanical stimuli experiencedby an ACL in vivo.

[0155] Various factors will influence the amount of force that can betolerated by the fiber construct (e.g., fiber construct composition,cell density). Fiber construct strength is expected to change throughthe course of tissue development. Therefore, applied mechanical forcesor strains will increase, decrease or remain constant in magnitude,duration, frequency and variety over the period of ligament generation,to appropriately correspond to fiber construct strength at the time ofapplication.

[0156] When producing an ACL, the more accurate the intensity andcombination of stimuli applied to the fiber construct during tissuedevelopment, the more the resulting ligament will resemble a native ACL.Two issues must be considered regarding the natural function of the ACLwhen devising the in vitro mechanical force regimen that closely mimicsthe in vivo environment: (1) the different types of motion experiencedby the ACL and the responses of the ACL to knee joint movements and (2)the extent of the mechanical stresses experienced by the ligament.Specific combinations of mechanical stimuli are generated from thenatural motions of the knee structure and transmitted to the native ACL.

[0157] To briefly describe the motions of the knee, the connection ofthe tibia and femur by the ACL provides six degrees of freedom whenconsidering the motions of the two bones relative to each other. Thetibia can move in three directions and can rotate relative to the axesfor each of these three directions. The knee is restricted fromachieving the full ranges of these six degrees of freedom due to thepresence of ligaments and capular fibers and the knee surfacesthemselves (Biden et al., “Experimental Methods Used to Evaluate KneeLigament Function,” Knee Ligaments: Structure, Function, Injury andRepair, Ed. D. Daniel et al., Raven Press, pp.135-151, 1990). Smalltranslational movements are also possible. The attachment sites of theACL are responsible for its stabilizing roles in the knee joint. The ACLfunctions as a primary stabilizer of anterior-tibial translation, and asa secondary stabilizer of valgus-varus angulation, and tibial rotation(Shoemaker et al., “The Limits of Knee Motion,” Knee Ligaments:Structure, Function, Injury and Repair, Ed. D. Daniel et al., RavenPress, pp.1534-161, 1990). Therefore, the ACL is responsible forstabilizing the knee in three of the six possible degrees of freedom. Asa result, the ACL has developed a specific fiber organization andoverall structure to perform these stabilizing functions. Theseconditions are simulated in vitro to produce a tissue with similarstructure and fiber organization.

[0158] The extent of mechanical stresses experienced by the ACL can besimilarly summarized. The ACL undergoes cyclic loads of about 400 Nbetween one and two million cycles per year (Chen et al., J. Biomed.Mat. Res. 14: 567-586, 1980). Also considered are linear stiffness (˜182N/mm), ultimate deformation (100% of ACL) and energy absorbed at failure(12.8 N-m) (Woo et al., The tensile properties of human anteriorcruciate ligament (ACL) and ACL graft tissues, Knee Ligaments:Structure, Function, Injury and Repair, Ed. D. Daniel et al. RavenPress, pp.279-289, 1990) when developing an ACL surgical replacement.

[0159] The examples section, below, details the production of aprototype bioengineered anterior cruciate ligament (ACL) ex vivo.Mechanical forces mimicking a subset of the mechanical stimuliexperienced by a native ACL in vivo (rotational deformation and lineardeformation) were applied in combination, and the resulting ligamentthat was formed was studied to determine the effects of the appliedforces on tissue development. Exposure of the developing ligament tophysiological loading during in vitro formation induced the cells toadopt a defined orientation along the axes of load, and to generateextracellular matrices along the axes as well. These results indicatethat the incorporation of complex multi-dimensional mechanical forcesinto the regime to produce a more complex network of load axes thatmimics the environment of the native ACL will produce a bioengineeredligament that more closely resembles a native ACL.

[0160] The different mechanical forces that may be applied include,without limitation, tension, compression, torsion, and shear. Theseforces are applied in combinations that simulate forces experienced byan ACL in the course of natural knee joint movements and function. Thesemovements include, without limitation, knee joint extension and flexionas defined in the coronal and sagittal planes, and knee joint flexion.Optimally, the combination of forces applied mimics the mechanicalstimuli experienced by an anterior cruciate ligament in vivo asaccurately as is experimentally possible. Varying the specific regimenof force application through the course of ligament generation isexpected to influence the rate and outcome of tissue development, withoptimal conditions to be determined empirically. Potential variables inthe regimen include, without limitation: (1) strain rate, (2) percentstrain, (3) type of strain (e.g., translation and rotation), (4)frequency, (5) number of cycles within a given regime, (6) number ofdifferent regimes, (7) duration at extreme points of ligamentdeformation, (8) force levels, and (9) different force combinations. Awide variety of variations exist. The regimen of mechanical forcesapplied can produce helically organized fibers similar to those of thenative ligament, described below.

[0161] The fiber bundles of a native ligament are arranged into ahelical organization. The mode of attachment and the need for the kneejoint to rotate ˜140° of flexion has resulted in the native ACLinheriting a 90° twist and with the peripheral fiber bundles developinga helical organization. This unique biomechanical feature allows the ACLto sustain extremely high loading. In the functional ACL, this helicalorganization of fibers allows anterior-posterior and posterior-anteriorfibers to remain relatively isometric in respect to one another for alldegrees of flexion, thus load can be equally distributed to all fiberbundles at any degree of knee joint flexion, stabilizing the kneethroughout all ranges of joint motion. Mechanical forces that simulate acombination of knee joint flexion and knee joint extension can beapplied to the developing ligament to produce an engineered ACL thatpossesses this same helical organization. The mechanical apparatus usedin the experiments presented in the examples, below, provides controlover strain and strain rates (both translational and rotational). Themechanical apparatus will monitor the actual load experienced-by thegrowing ligaments, serving to ‘teach’ the ligaments over time throughmonitoring and increasing the loading regimes.

[0162] Another aspect of this disclosure relates to the bioengineeredanterior cruciate ligament produced by the above-described methods. Thebioengineered ligament produced by these methods is characterized bycellular orientation and/or a fiber construct crimp pattern in thedirection of the mechanical forces applied during generation. Theligament is also characterized by the production/presence ofextracellular fiber construct components (e.g., collagen type I and typeIII, fibronectin, and tenascin-C proteins) along the axis of mechanicalload experienced during culture. The ligament fiber bundles can bearranged into a helical organization, as discussed above.

[0163] The above methods using the novel silk-fiber-based fiberconstruct are not limited to the production of an ACL, but can also beused to produce other ligaments and tendons found in the knee (e.g.,posterior cruciate ligament) or other parts of the body (e.g., hand,wrist, ankle, elbow, jaw and shoulder), such as for example, but notlimited to posterior cruciate ligament, rotator cuff tendons, medialcollateral ligament of the elbow and knee, flexor tendons of the hand,lateral ligaments of the ankle and tendons and ligaments of the jaw ortemporomandibular joint. All moveable joints in a human body havespecialized ligaments that connect the articular extremities of thebones in the joint. Each ligament in the body has a specific structureand organization that is dictated by its function and environment. Thevarious ligaments of the body, their locations and functions are listedin Anatomy, Descriptive and Surgical (Gray, H., Eds. Pick, T. P.,Howden, R., Bounty Books, New York, 1977), the pertinent contents ofwhich are incorporated herein by reference. By determining the physicalstimuli experienced by a given ligament or tendon, and incorporatingforces which mimic these stimuli, the above-described method forproducing an ACL ex vivo can be adapted to produce bioengineeredligaments and tendons ex vivo that simulates any ligament or tendon inthe body.

[0164] The specific type of ligament or tendon to be produced ispredetermined prior to tissue generation since several aspects of themethod vary with the specific conditions experienced in vivo by thenative ligament or tendon. The mechanical forces to which the developingligament or tendon is subjected during cell culture are determined forthe particular ligament or tendon type being cultivated. The specificconditions can be determined by studying the native ligament or tendonand its environment and function. One or more mechanical forcesexperienced by the ligament or tendon in vivo are applied to the fiberconstruct during culture of the cells in the fiber construct. Theskilled practitioner will recognize that a ligament or tendon that issuperior to those currently available can be produced by the applicationof a subset of forces experienced by the native ligament or tendon.However, optimally, the full range of in vivo forces will be applied tothe fiber construct in the appropriate magnitudes and combinations toproduce a final product that most closely resembles the native ligamentor tendon. These forces include, without limitation, the forcesdescribed above for the production of an ACL. Because the mechanicalforces applied vary with ligament or tendon type, and the final size ofthe ligament or tendon will be influenced by the anchors used, optimalanchor composition, size and fiber construct attachment sites are to bedetermined for each type of ligament or tendon by the skilledpractitioner. The type of cells seeded on the fiber construct isobviously determined based on the type of ligament or tendon to beproduced.

[0165] Other tissue types can be produced ex vivo using methods similarto those described above for the generation of ligaments or tendons exvivo. The above-described methods can also be applied to produce a rangeof engineered tissue products that involve mechanical deformation as amajor part of their function, such as muscle (e.g., smooth muscle,skeletal muscle, cardiac muscle), bone, cartilage, vertebral discs, andsome types of blood vessels. Bone marrow stromal cells possess theability to differentiate into these as well as other tissues. Thegeometry of the silk-based fiber construct or composite fiber constructcan easily be adapted to the correct anatomical geometricalconfiguration of the desired tissue type. For example, silk fibroinfibers can be reformed in a cylindrical tube to recreate arteries.

[0166] The results presented in the examples, below, indicate thatgrowth in an environment that mimics the specific mechanical environmentof a given tissue type will induce the appropriate cell differentiationto produce a bioengineered tissue that significantly resembles nativetissue. The ranges and types of mechanical deformation of the fiberconstruct can be extended to produce a wide range of tissue structuralorganization. The cell culture environment can reflect the in vivoenvironment experienced by the native tissue and the cells it contains,throughout the course of embryonic development to mature function of thecells within the native tissue, as accurately as possible. Factors toconsider when designing specific culture conditions to produce a giventissue include, without limitation, the fiber construct composition, themethod of cell immobilization, the anchoring method of the fiberconstruct or tissue, the specific forces applied, and the cell culturemedium. The specific regimen of mechanical stimulation depends upon thetissue type to be produced, and is established by varying theapplication of mechanical forces (e.g., tension only, torsion only,combination of tension and torsion, with and without shear, etc.), theforce amplitude (e.g., angle or elongation), the frequency and durationof the application, and the duration of the periods of stimulation andrest.

[0167] The method for producing the specific tissue type ex vivo is anadaptation of the above-described method for producing an ACL.Components involved include pluripotent cells, a three-dimensional fiberconstruct to which cells can adhere, and a plurality of anchors thathave a face suitable for fiber construct attachment. The pluripotentcells (such as bone marrow stromal cells) are seeded in the threedimensional fiber construct by means to uniformly immobilize the cellswithin the fiber construct. The number of cells seeded is also notviewed as limiting, however, seeding the fiber construct with a highdensity of cells may accelerate tissue generation.

[0168] The specific forces applied are to be determined for each tissuetype produced through examination of native tissue and the mechanicalstimuli experienced in vivo. A given tissue type experiencescharacteristic forces that are dictated by location and function of thetissue within the body. For instance, cartilage is known to experience acombination of shear and compression/tension in vivo; bone experiencescompression.

[0169] Additional stimuli (e.g., chemical stimuli, electromagneticstimuli) can also be incorporated into the above-described methods forproducing bioengineered ligaments, tendons and other tissues. Celldifferentiation is known to be influenced by chemical stimuli from theenvironment, often produced by surrounding cells, such as secretedgrowth or differentiation factors, cell-cell contact, chemicalgradients, and specific pH levels, to name a few. Other more uniquestimuli are experienced by more specialized types of tissues (e.g., theelectrical stimulation of cardiac muscle). The application of suchtissue specific stimuli (e.g., 1-10 ng/ml transforming growth factorbeta-1 (TGF-β1) independently or in concert with the appropriatemechanical forces is expected to facilitate differentiation of the cellsinto a tissue that more closely approximates the specific naturaltissue.

[0170] Tissues produced by the above-described methods provide anunlimited pool of tissue equivalents for surgical implantation into acompatible recipient, particularly for replacement or repair of damagedtissue. Engineered tissues may also be utilized for in vitro studies ofnormal or pathological tissue function, e.g., for in vitro testing ofcell- and tissue-level responses to molecular, mechanical, or geneticmanipulations. For example, tissues based on normal or transfected cellscan be used to assess tissue responses to biochemical or mechanicalstimuli, identify the functions of specific genes or gene products thatcan be either over-expressed or knocked-out, or to study the effects ofpharmacological agents. Such studies will likely provide more insightinto ligament, tendon and tissue development, normal and pathologicalfunction, and eventually lead toward fully functional tissue engineeredreplacements, based in part on already established tissue engineeringapproaches, new insights into cell differentiation and tissuedevelopment, and the use of mechanical regulatory signals in conjunctionwith cell-derived and exogenous biochemical factors to improvestructural and functional tissue properties.

[0171] The production of engineered tissues, such as ligaments andtendons, also has the potential for applications such as harvesting bonemarrow stromal cells from individuals at high risk for tissue injury(e.g., ACL rupture) prior to injury. These cells could be either storeduntil needed or seeded into the appropriate fiber construct and culturedand differentiated in vitro under mechanical stimuli to produce avariety of bioengineered prosthetic tissues to be held in reserve untilneeded by the donor. The use of bioengineered living tissue prostheticsthat better match the biological environment in vivo and that providethe required physiological loading to sustain, for example, the dynamicequilibrium of a normal, fully functional ligament should reducerehabilitation time for a recipient of a prosthesis from months toweeks, particularly if the tissue is pre-grown and stored. Benefitsinclude a more rapid regain of functional activity, shorter hospitalstays, and fewer problems with tissue rejections and failures.

[0172] Additional aspects of this invention are further exemplified inthe following examples. It will be apparent to those skilled in the artthat many modifications, both to the materials and methods, may bepracticed without departing from the invention.

[0173] In a first example, raw Bombyx mori silkworm fibers, shown inFIG. 1A, were extracted to remove sericin, the glue-like protein coatingthe native silk fibroin (see FIGS. 1A-C). The appropriate number offibers per group were arranged in parallel and extracted in an aqueoussolution of 0.02 M Na2CO3 and 0.3% (w/v) IVORY soap solution for 60minutes at 90° C., then rinsed thoroughly with water to extract theglue-like sericin proteins.

[0174] Costello's equation for a three-strand, helical rope geometry wasderived to predict mechanical properties of the silk-fiber-basedconstruct. The derived model is a series of equations that whencombined, take into account extracted silk fiber material properties anddesired fiber construct geometrical hierarchy to compute the overallstrength and stiffness of the fiber construct as a function of pitchangle for a given level of geometrical hierarchy.

[0175] The material properties of a single silk fiber include fiberdiameter, modulus of elasticity, Poisson's ratio, and the ultimatetensile strength (UTS). Geometrical hierarchy may be defined as thenumber of twisting levels in a given fiber construct level. Each level(e.g., group, bundle, strand, cord, ligament) is further defined by thenumber of groups of fibers twisted about each other and the number offibers in each group of the first level twisted where the first level isdefine as a group, the second level as a bundle, the third as a strandand the fourth as a cord, the fifth as the ligament.

[0176] The model assumes that each group of multiple fibers act as asingle fiber with an effective radius determined by the number ofindividual fibers and their inherent radius, i.e., the model discountsfriction between the individual fibers due to its limited role in givena relatively high pitch angle.

[0177] Two applicable geometries (Matrix 1 and Matrix 2) of the manyfiber construct geometrical configurations (see Table 10, supra)computed to yield mechanical properties mimicking those of a native ACLwere derived for more detailed analysis. A six-cord construct wasselected for use as the ACL replacement. Matrix configurations are asfollows: Matrix 1: 1 ACL prosthesis=6 parallel cords; 1 cord=3 twistedstrands (3 twists/cm); 1 strand=6 twisted bundles (3 twists/cm); 1bundle=30 parallel washed fibers; and Matrix 2: 1 ACL matrix=6 parallelcords; 1 cord=3 twisted strands (2 twists/cm); 1 strand=3 twistedbundles (2.5 twists/cm); 1 bundle=3 groups (3 twists/cm); 1 group=15parallel extracted silk fibroin fibers. The number of fibers andgeometries were selected such that the silk prostheses are similar tothe ACL biomechanical properties in UTS, linear stiffness, yield pointand % elongation at break (see Table 10, supra), thus serving as a solidstarting point for the development of a tissue engineered ACL.

[0178] Mechanical properties of the silk fibroin were characterizedusing a servohydraulic Instron 8511 tension/compression system withFast-Track software (Instron Corp., Canton, Mass., USA) (see FIG. 1D).Single pull-to-failure and fatigue analyses were performed on singlesilk fibers, extracted fibroin and organized cords. Fibers and fibroinwere organized in both the parallel helical geometries of Matrix 1 (seeFIG. 2C) and of Matrix 2 (see FIG. 2D) for characterization. Single pullto failure testing was performed at a strain rate of 100%/sec; forceelongation histograms were generated and data analyzed using InstronSeries 1X software. Both Matrix 1 and Matrix 2 yielded similarmechanical and fatigue properties to the ACL in UTS, linear stiffness,yield point and percent elongation at break (see Table 10 and FIGS.3A-D).

[0179] Fatigue analyses were performed using a servohydraulic Instron8511 tension/compression system with Wavemaker software on single cordsof both Matrix 1 and Matrix 2. Data was extrapolated to represent the6-cord ACL prostheses, which is shown in FIGS. 3B and 3D. Cord ends wereembedded in an epoxy mold to generate a 3-cm-long construct betweenanchors. Cycles to failure at UTS's of 1,680 N and 1,200 N (n=5 for eachload) for Matrix 1 (see FIG. 3B) and at UTS's of 2280 N, 2100 N and 1800N loads (n=3 for each load) for Matrix 2 (see FIG. 3D) were determinedusing a H-sine wave function at 1 Hz generated by Wavemaker 32 version6.6 software (Instron Corp.). Fatigue testing was conducted in a neutralphosphate buffered saline (PBS) solution at room temperature.

[0180] Complete sericin removal was observed after 60 min at 90° C. asdetermined by SEM (see FIGS. 1A-C). Removal of sericin from silk fibersaltered the ultrastructure of the fibers, resulting in a smoother fibersurface, and the underlying silk fibroin was revealed (shown in FIGS.1A-C), with average diameter ranging between 20-40 μm. The fibroinexhibited a significant 15.2% decrease in ultimate tensile strength(1.033±0.042 N/fiber to 0.876±0.1 N/fiber) (p<0.05, paired Studentst-test) (see FIG. 1D). The mechanical properties of the optimized silkmatrices (see FIG. 2A-D & FIG. 3A-D) are summarized in Table 11 aboveand in FIG. 3A (for Matrix 1) and in FIG. 3C (for Matrix 2). It isevident from these results that the optimized silk matrices exhibitedvalues comparable to those of native ACL, which have been reported tohave an average ultimate tensile strength (UTS) of ˜2100 N, stiffness of˜250 N/nm, yield point ˜2100 N and 33% elongation at break (See Woo, SL-Y, et al., The Tensile Properties of Human Anterior Cruciate Ligament(ACL) and ACL Graft Tissue in Knee Ligaments: Structure, Function,Injury and Repair, 279-289, Ed. D. Daniel et al., Raven Press 1990).

[0181] Regression analysis of fiber construct fatigue data, shown inFIG. 3B for Matrix 1 and in FIG. 3D for Matrix 2, when extrapolated tophysiological load levels (400 N) predict the number of cycles tofailure in vivo, indicate a fiber construct life of 3.3 million cyclesfor Matrix 1 and a life of greater than 10 million cycles for Matrix 2.The helical fiber construct design utilizing washed silk fibers resultedin a fiber construct with physiologically equivalent structuralproperties, confirming its suitability as a scaffold for ligament tissueengineering.

[0182] In another example involving cell isolation and culture, bonemarrow stromal cells (BMSC), pluripotent cells capable ofdifferentiating into osteogenic, chondrogenic, tendonogenic, adipogenicand myogenic lineages, were chosen since the formation of theappropriate conditions can direct their differentiation into the desiredligament fibroblast cell line (Markolfet al., J. Bone Joint Surg. 71A:887-893, 1989; Caplan et al., Mesenchymal stem cells and tissue repair,The Anterior Cruciate Ligament: Current and Future Concepts, Ed. D. W.Jackson et al., Raven Press, Ltd, New York, 1993; Young et al., J.Orthopaedic Res. 16: 406-413, 1998).

[0183] Human BMSCs were isolated from bone marrow from the iliac crestof consenting donors at least 25 years of age by a commercial vendor(Cambrex, Walkersville, Md.). Twenty-two milliliters of human marrow wasaseptically aspirated into a 25 ml syringe containing three millilitersof heparinized (1000 units per milliliter) saline solution. Theheparinized marrow solution was shipped overnight on ice to thelaboratory for bone marrow stromal cells isolation and culture. Uponarrival from the vendor, the twenty-five milliliter aspirates wereresuspended in Dulbecco's Modified Eagle Medium (DMEM) supplemented with10% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, 100 U/mlpenicillin, 100 mg/L streptomycin (P/S), and 1 ng/ml basic fibroblastgrowth factor (bFGF) (Life Technologies, Rockville, Md.) and plated at8-10 microliters of aspirate/cm2 in tissue culture flasks. Fresh mediumwas added to the marrow aspirates twice a week for up to nine days ofculture. BMSCs were selected based on their ability to adhere to thetissue culture plastic; non-adherent hematopoietic cells were removedduring medium replacement after 9-12 days in culture. Medium was changedtwice per week thereafter. When primary BMSC became near confluent(12-14 days), they were detached using 0.25% trypsin/1 mM EDTA andreplated at 5×103 cells/cm2. First passage (P1) hBMSCs were trypsinizedand frozen in 8% DMSO/10% FBS/DMEM for future use.

[0184] Frozen P1 hBMSCs were defrosted, replated at 5×103 cells/cm2(P2), trypsinized when near confluency, and used for fiber constructseeding. Sterilized (ethylene oxide) silk matrices (specifically, singlecords of Matrices 1 and 2, bundles of 30 parallel extracted silk fibers,and helical ropes of collage fibers) were seeded with cells incustomized seeding chambers (1 ml total volume) machined in Teflonblocks to minimize cell-medium volume and increase cell-fiber constructcontact. Seeded matrices, following a 4 hour incubation period with thecell slurry (3.3×106 BMSCs/ml) were transferred into a petri dishcontaining an appropriate amount of cell culture medium for the durationof the experiments.

[0185] To determine the degradation rate of the silk fibroin, ultimatetensile strength (UTS) was measured as a function of cultivation periodin physiological growth conditions, i.e., in cell culture medium. Groupsof 30 parallel silk fibers 3 cm in length were extracted, seeded withhBMSCs, and cultured on the fibroin over 21 days at 37° C. and 5% CO2.Non-seeded control groups were cultured in parallel. Silk fibroin UTSwas determined as a function of culture duration for seeded andnon-seeded groups.

[0186] The response of bone marrow stromal cells to the silk fiberconstruct was also examined.

[0187] BMSCs readily attached and grew on the silk and collagen matricesafter 1 day in culture (See FIG. 7A-C and FIG. 16A), and formed cellularextensions to bridge neighboring fibers. As shown in FIG. 7D and FIG.16B, a uniform cells sheet covering the construct was observed at 14 and21 days of culture, respectively. MTT analysis confirmed complete fiberconstruct coverage by seeded BMSCs after 14 days in culture (see FIG.8A-B). Total DNA quantification of cells grown on Matrix 1 (see FIG. 9A)and Matrix 2 (see FIG. 9B) confirmed that BMSCs proliferated and grew onthe silk construct with the highest amount of DNA measured after 21 and14 days, respectively, in culture.

[0188] Both BMSC-seeded or non-seeded extracted control silk fibroingroups of 30 fibers, maintained their mechanical integrity as a functionof culture period over 21 days (see FIG. 10).

[0189] RT-PCR analysis of BMSCs seeded on cords of Matrix 2 indicatedthat both collagen I & III were upregulated over 14 days in culture(FIG. 14). Collagen type II and bone sialoprotein (as indicators ofcartilage and bone specific differentiation, respectively) were eithernot detectable or negligibly expressed over the cultivation period.Real-time quantitative RT-PCR at 14 days yielded a transcript ratio ofcollagen I to collagen III, normalized to GAPDH, of 8.9:1 (see FIG. 17).The high ratio of collagen I to collagen III indicates that the responseis not wound healing or scar tissue formation (as is observed with highlevels of collagen-type III), but rather ligament specific; the relativeratio of collagen I to collagen III in a native ACL is ˜6.6:1 (Amiel etal., Knee Ligaments: Structure, Function, Injury, and Repair, 1990).

[0190] Additionally, studies are conducted to provide insight into theinfluence of directed multi-dimensional mechanical stimulation onligament formation from bone marrow stromal cells in the bioreactorsystem. The bioreactor is capable of applying independent but concurrentcyclic multi-dimensional strains (e.g., translation, rotation) to thedeveloping ligaments. After a 7 to 14 day static rest period (time postseeding), the rotational and translation strain rates and linear androtational deformation are kept constant for 1 to 4 weeks. Translationalstrain (3.3%-10%, 1-3 mm) and rotational strain (25%, 90°) areconcurrently applied at a frequency of 0.0167 Hz (one full cycle ofstress and relaxation per minute) to the silk-based matrices seeded withBMSCs; an otherwise identical set of bioreactors with seeded matriceswithout mechanical loading serve as controls. The ligaments are exposedto the constant cyclic strains for the duration of the experiment days.

[0191] Following the culture period, ligament samples, both themechanically challenged as well as the controls (static) arecharacterized for: (1) general histomorphological appearance (by visualinspection); (2) cell distribution (image processing of histological andMTT stained sections); (3) cell morphology and orientation (histologicalanalysis); and (4) the production of tissue specific markers (RT-PCR,immunostaining).

[0192] Mechanical stimulation markedly affects the morphology andorganization of the BMSCs and newly developed extracellular fiberconstruct, the distribution of cells along the fiber construct, and theupregulation of a ligament-specific differentiation cascade; BMSCs alignalong the long axis of the fiber, take on a spheroid morphology similarto ligament/tendon fibroblasts and upregulate ligament/tendon specificmarkers. Newly formed extracellular fiber construct (i.e., thecomposition of proteins produced by the cells) is expected to alignalong the lines of load as well as the long axis of the fiber construct.Directed mechanical stimulation is expected to enhance ligamentdevelopment and formation in vitro in a bioreactor resulting from BMSCsseeded on the novel silk-based fiber construct. The longitudinalorientation of cells and newly formed fiber construct is similar toligament fibroblasts found within an ACL in vivo (Woods et al., Amer. J.Sports Med. 19: 48-55, 1991). Furthermore, mechanical stimulationmaintains the correct expression ratio between collagen type Itranscripts and collagen type III transcripts (e.g., greater than 7:1)indicating the presence of newly formed ligament tissue versus scartissue formation. The above results will indicate that the mechanicalapparatus and bioreactor system provide a suitable environment (e.g.,multi-dimensional strains) for in vitro formation of tissue engineeredligaments starting from bone marrow stromal cells and the novelsilk-based fiber construct.

[0193] The culture conditions used in these preliminary experiments canbe further expanded to more accurately reflect the physiologicalenvironment of a ligament (e.g., increasing the different types ofmechanical forces) for the in vitro creation of functional equivalentsof native ACL for potential clinical use. These methods are not limitedto the generation of a bioengineered ACL. By applying the appropriatemagnitude and variety of forces experienced in vivo, any type ofligament in the body as well as other types of tissue can be produced exvivo by the methods of this disclosure.

[0194] Other embodiments are within the following claims. TABLE 1Ultimate tensile strength and stiffness (N/mm given a 3 cm long sample)as a function of sericin extraction from a 10-fiber silkworm silk yarnwith 0 twists per inch (i.e., parallel) and (i) temperature and (ii)time. Repeat samples were processed two years after initial samples withno significant change in properties. N = 5 for all samples. # of UTSStiff UTS/ Stiffness/fiber Yarn fibers Temp Time (N) stdev (N/mm) stdevfiber (N) (N/mm) 10(0) 10 RT 60 min 10.74 0.83 6.77 0.65 1.07 0.68 10(0)10 RT 60 min (repeat) 10.83 0.28 6.36 0.14 1.08 0.64 10(0) 10 33 C. 60min 10.44 0.17 6.68 0.55 1.04 0.67 10(0) 10 37 C. 60 min 9.60 0.84 6.090.59 0.96 0.61 10(0) 10 37 C. 60 min (repeat) 9.54 0.74 5.81 0.67 0.950.58 10(0) 10 90 C. 15 min 9.22 0.55 4.87 0.62 0.92 0.49 10(0) 10 90 C.30 min 8.29 0.19 4.91 0.33 0.83 0.49 10(0) 10 90 C. 60 min 8.60 0.614.04 0.87 0.86 0.40 10(0) 10 90 C. 60 min (repeat) 8.65 0.67 4.55 0.690.87 0.46 10(0) 10 94 C. 60 min 7.92 0.51 2.42 0.33 0.79 0.24 9(12s) ×3(9z) 27 non-extracted 24.50 0.38 8.00 0.49 0.91 0.30 9(12s) × 3(9z) 2790 C. 60 min 21.88 0.18 7.38 0.34 0.81 0.27  9(6s) × 3(3z) 27non-extracted 24.94 0.57 9.51 0.57 0.92 0.35  9(6s) × 3(3z) 27 90 C. 60min 21.36 0.40 7.95 1.00 0.79 0.29 9(12s) × 3(6z) 27 non-extracted 24.690.65 9.08 0.56 0.91 0.34 9(12s) × 3(6z) 27 90 C. 60 min 21.80 0.47 7.480.97 0.81 0.28

[0195] TABLE 2 Mass loss as a function of sericin extraction. +/−0.43%standard deviation of an N = 5, reflects the greatest accuracy that canbe achieved when confirming sericin removal, i.e., 0.87 or 1% error willalways be inherent to the methods used and a mass loss of about 24%represents substantially sericin free constructs. non-extractedextracted and % mass yarn and dried (mg) dried (mg) loss 9(12) × 3(6)57.6 43.6 24.31 9(12) × 3(6) 58.3 43.9 24.70 9(12) × 3(6) 57.0 42.924.74 9(12) × 3(6) 57.2 42.7 25.35 average 57.53 43.28 24.77 stdev 0.570.57 0.43

[0196] TABLE 3 Illustrates the change in mass as a function of a secondsericin extraction. Correlated to FIG. 1E-1G, less than a 3% mass lossis likely indicative of fibroin mass loss due to mechanical damageduring the 2^(nd) extraction. mass after 1x mass after 2x extraction,extracted, dried % mass yarn dried (mg) (mg) loss 9(12) × 3(6) 42.5 41.71.88 9(12) × 3(6) 43.1 42 2.55 9(12) × 3(6) 43.1 42.1 2.32 9(12) × 3(6)42.5 41.7 1.88 9(12) × 3(6) 42.6 42.4 0.47 9(12) × 3(6) 43.7 42.4 2.979(12) × 3(6) 43.4 42.9 1.15 9(12) × 3(6) 43.7 43.1 1.37 9(12) × 3(6) 4443.2 1.82 average 43.18 42.39 1.82 stdev 0.56 0.57 0.76

[0197] TABLE 4 # of Total # UTS UTS % % Stiffness Stiffness UTSStiffness Ply levels of of average stdev Elong Elong avg stdev per perGeometry Method Condition plying Fibers (N) (N) average stdev (N/mm)(N/mm) fiber fiber 1(0) × 3(10) cable extracted 2 3 1.98 0.05 10.42 1.632.17 0.51 0.66 0.72 1(0) × 4(10) cable extracted 2 4 2.86 0.14 11.981.54 2.08 0.31 0.72 0.52 3(0) × 3(3) cable extracted 2 9 6.72 0.17 12.300.72 4.54 0.16 0.75 0.50 1(0) × 3(10) × 3(9) cable extracted 3 9 6.860.23 13.11 1.45 4.06 0.36 0.76 0.45 2(0) × 6(11) cable 2 12 7.97 0.2610.05 0.91 0.66 4(6) × 3(3) twist non-extracted 2 12 10.17 0.18 19.861.16 0.85 1(0) × 3(10) × 4(9) cable extracted 3 12 9.29 0.19 14.07 0.985.10 0.31 0.77 0.43 1(0) × 4(11) × 3(11) twist extracted 3 12 9.70 0.1412.56 1.03 7.60 0.33 0.81 0.63 1(0) × 4(10) × 3(9) cable extracted 3 128.78 0.17 14.25 1.09 5.10 0.32 0.73 0.43 15(textured) texturednon-extracted, 1 15 10.62 0.68 10.76 1.70 4.75 0.30 0.71 0.316 dry 30(0)parallel extracted, wet 1 30 20.24 1.46 26.32 3.51 1.14 0.15 0.67 0.03830(0) parallel incubated 21 1 30 19.73 2.10 20.70 6.03 0.66 days, wet30(0) parallel cell-seeded 21 1 30 20.53 1.02 29.68 7.08 0.68 days, wet2 fibers/carrier in an 8 braid extracted, dry 2 16 10.93 0.13 6.96 1.140.68 0.435 4 fibers/carrier in an 8 braid extracted, dry 2 32 24.60 0.2212.39 0.53 0.77 0.387  4(6) × 3(3) in 4 braid extracted, dry 3 48 37.670.18 22.38 0.98 0.78 carrier 15(0) × 3(12) cable dry 2 45 27.39 0.6231.68 1.35 4.63 0.49 0.61 0.102889 15(0) × 3(12) × 3(10) cablenon-extracted, 3 135 73.61 6.00 33.72 5.67 12.33 1.53 0.55 0.091333 1day after manufacturing 15(0) × 3(12) × 3(10) cable non-extracted, 3 13572.30 5.68 31.18 4.35 0.54 2 days after manufacturing 15(0) × 3(12) ×3(10) cable non-extracted, 3 135 70.74 2.97 29.50 4.47 0.52 3 days aftermanufacturing 15(0) × 3(12) × 3(10) cable non-extracted, 3 135 75.901.57 34.57 4.12 0.56 4 day after manufacturing 15(0) × 3(12) × 3(10)cable non-extracted, 3 135 71.91 5.71 36.72 3.75 0.53 5 days aftermanufacturing 15(0) × 3(12) × 3(10) cable non-extracted, 3 135 74.571.45 37.67 4.27 0.55 6 days after manufacturing 13(0) × 3(11) × cablenon-extracted, 4 351 189.01 14.00 45.87 3.72 0.54 3(10) × 3(0) dry 13(0)× 3(11) × cable non-extracted, 4 351 170.12 7.37 39.95 1.37 0.48 3(10) ×3(0) cycled 30× to pretension, dry

[0198] TABLE 5 Comparison of UTS and stiffness between wet (2 hrincubation in PBS at 37° C.) and dry mechanical testing conditions. N =5. Results show approximately a 17% drop in UTS as a function of testingwet. # of Yarn Test UTS UTS Stiffness Stiffness UTS/fiberStiffness/fiber Yarn Fibers Conditions (N) stdev (N/mm) stdev (N) (N/mm)9(12s) × 3(9z) 27 extracted - dry 21.88 0.18 7.38 0.34 0.81 0.27 9(12s)× 3(9z) 27 extracted - wet 18.52 0.25 2.56 0.31 0.69 0.09  9(6s) × 3(3z)27 extracted - dry 21.36 0.40 7.95 1.00 0.79 0.29  9(6s) × 3(3z) 27extracted - wet 17.94 0.30 2.40 0.28 0.66 0.09 9(12s) × 3(6z) 27extracted - dry 21.80 0.47 7.48 0.97 0.81 0.28 9(12s) × 3(6z) 27extracted - wet 18.74 0.22 2.57 0.11 0.69 0.10  12(0) × 3(10s) 36extracted - dry 30.73 0.46 16.24 0.66 0.85 0.45  12(0) × 3(10s) 36extracted - wet 25.93 0.29 6.68 0.70 0.72 0.19  4(0) × 3(10s) × 3(9z) 36extracted - dry 30.07 0.35 15.49 1.06 0.84 0.43  4(0) × 3(10s) × 3(9z)36 extracted - wet 22.55 0.66 7.63 1.00 0.63 0.21

[0199] TABLE 6 Effect of TPI on UTS and Stiffness. N = 5 UTS StiffnessUTS/fiber Stiffness/fiber Yarn TPI (N) stdev (N/mm) stdev (N) (N/mm)12(0) × 3(2) 2 23.27 0.28 6.86 0.60 0.65 0.19 12(0) × 3(4) 4 24.69 0.317.61 1.17 0.69 0.21 12(0) × 3(6) 6 25.44 0.42 6.51 1.35 0.71 0.18 12(0)× 3(8) 8 25.21 0.23 5.80 0.67 0.70 0.16 12(0) × 3(10) 10 25.94 0.24 6.450.77 0.72 0.18 12(0) × 3(12) 12 25.87 0.19 6.01 0.69 0.72 0.17 12(0) ×3(14) 14 22.21 0.58 5.63 0.71 0.62 0.16

[0200] TABLE 7 Additional tpi data to verify that up to 30 tpi can beused without causing damage to the yarn that would result in a dramaticdecrease in UTS and stiffness; note, all matrices (N = 5 per group) weretwisted. # of UTS stdev Stiffness stdev UTS/fiber Stiffness/fiber Yarnfibers (N) (N) (N/mm) (N/mm) (N) (N/mm) Conditions 1(30) × 6(20) ×3(4.5) 18 10.92 0.44 1.21 0.02 0.61 0.07 non-extracted, wet 1(30) ×6(20) × 3(10) 18 11.48 0.37 1.25 0.06 0.64 0.07 non-extracted, wet 1(30)× 6(6) 6 3.83 0.24 0.37 0.04 0.64 0.06 non-extracted, wet 15(20) 1513.19 0.27 6.03 0.67 0.88 0.40 extracted, dry

[0201] TABLE 8 Effect of yarn hierarchy on mechanical properties (i.e.the number of levels and the number of fibers per level cansignificantly influence yarn and fabric outcomes. # of Total # UTS % %Stiffness Stiffness UTS Stiffness levels of of UTS stdev Elong Elong avgstdev per per Geometry Condition plying Fibers (N) (N) average stdev(N/mm) (N/mm) fiber fiber  1(0) × 3(10) extracted 2 3 1.98 0.05 10.421.63 2.17 0.51 0.66 0.72  1(0) × 3(10) × 3(9) extracted 3 9 6.86 0.2313.11 1.45 4.06 0.36 0.76 0.45  1(0) × 3(10) × 4(9) extracted 3 12 9.290.19 14.07 0.98 5.10 0.31 0.77 0.43  1(0) × 4(10) extracted 2 4 2.860.14 11.98 1.54 2.08 0.31 0.72 0.52  1(0) × 4(10) × 3(9) extracted 3 128.78 0.17 14.25 1.09 5.10 0.32 0.73 0.43 15(0) × 3(12) non-extracted dry2 45 27.39 0.62 31.68 1.35 4.63 0.49 0.61 0.10 15(0) × 3(12) × 3(10)non-extracted dry 3 135 73.61 6.00 33.72 5.67 12.33 1.53 0.55 0.09

[0202] TABLE 9 Surface modification (RGD and ETO gas sterilization)effects on extracted silk matrix mechanical properties; PBS was used asa negative control during modification treatments. # of SurfaceModification/ UTS Stiffness UTS/fiber Stiffness/fiber Yarn fibersSterilization (N) stdev (N/mm) stdev (N) (N/mm)  12(0) × 3(10s)  36Non-treated 25.94 0.24 6.45 0.77 0.72 0.18  12(0) × 3(10s)  36 RGD 23.822.10 3.79 2.06 0.66 0.11  12(0) × 3(10s) × 3(9z) 108 Non-treated 48.894.84 9.22 0.84 0.45 0.09  12(0) × 3(10s) × 3(9z) 108 RGD 55.28 3.28 8.170.81 0.51 0.08 4(11s) × 3(11z) ×  36 ETO 18.72 0.45 5.52 0.42 0.52 0.153(10s) 4(11s) × 3(11z) ×  36 RGD + ETO 19.30 0.62 4.67 0.3 0.54 0.133(10s)

[0203] TABLE 10 UTS Stiffness Yield Pt. Elongation (N) (N/mm) (N) (%)Silk matrix 1 2337 +/− 72 354 +/− 26   1262 +/− 36 38.6 +/− 2.4 SilkMatrix 2 3407 +/− 63 580 +/− 40   1647 +/− 214   29 +/− 4 Human ACL 2160+/− 242 +/− 28 ˜1200 ˜26-32%  157

[0204] TABLE 11 Twisting Level Matrix Matrix Matrix Matrix Matrix MatrixMatrix (# of twists/cm) 1 2 3 4 5 6 7 # fibers per group  30  15 1300 180  20  10  15 (0) (0) (0) (0) (0) (0) (0) # groups per bundle   6   3  3   3   6   6   3 (3) (3) (2) (3.5) (3) (3) (3) # bundles per strand  3   6   1   3   3   3   3 (3) (2.5) (0) (2) (2) (2.5) (2.5) # strandsper cord   6   3 —   2   3   3   3 (0) (2.0) (0) (1) (2) (2) # cords perACL —   6 — —   3   6  12 (0) (0) (0) (0) UTS (N) 2337 3407 2780 23002500 2300 3400 Stiffness (N/mm)  354  580  300  350  550  500  550

1. A fabric comprising: a yarn, said yarn comprising one or moresericin-extracted fibroin fibers, said fibers being biocompatible andnon-randomly organized, wherein said yarn promotes ingrowth of cellsaround said fibroin fibers and is biodegradable.
 2. The fabric asrecited in claim 1, wherein the sericin-extracted fibroin fiberscomprises fibroin fibers obtained from Bombyx mori silkworm.
 3. Thefabric of claim 1, wherein the sericin-extracted fibroin fibers retaintheir native protein structure and have not been dissolved andreconstituted.
 4. The fabric of claim 1, wherein the fabric isnon-immunogenic.
 5. The fabric of claim 1, wherein the sericin-extractedfibroin fibers include less than 20% sericin by weight.
 6. The fabric ofclaim 1, wherein the sericin-extracted fibroin fibers include less than10% sericin by weight.
 7. The fabric of claim 1, wherein thesericin-extracted fibroin fibers include less than 1% sericin by weight.8. The fabric of claim 1, wherein the yarn has an ultimate tensilestrength of at least 0.52 N per fiber.
 9. The fabric of claim 8, whereinthe yarn has a stiffness between about 0.27 and about 0.5 N/mm perfiber.
 10. The fabric of claim 9, wherein the yarn retains 80% of itsUTS when tested wet.
 11. The fabric of claim 9, wherein the yarn has anelongation at break between about. 10% and about 50%.
 12. The fabric ofclaim 11, wherein the yarn has a fatigue life of at least 1 millioncycles at a load of about 20% of the yarn's ultimate tensile strength.13. The fabric of claim 1, wherein the yarn comprises parallel orintertwined sericin-extracted fibroin fibers.
 14. The yarn of claim 13,wherein said yarn comprises at least three aligned sericin-extractedfibroin fibers.
 15. The yarn of claim 14, wherein the alignedsericin-extracted fibroin fibers are intertwined.
 16. The yarn of claim15, wherein the yarn is a braid, textured yarn, twisted yarn, cabledyarn, and combinations thereof.
 17. The yarn of claim 16, wherein thealigned sericin-extracted fibroin fibers are twisted or cabled abouteach other at 0 to 11.8 twists per cm.
 18. The fabric of claim 1,further comprising a yarn having a single-level hierarchicalorganization, said single-level hierarchical organization comprising agroup of parallel or intertwined yarns.
 19. The fabric of claim 1,further comprising a yarn having a two-level hierarchical organization,said two-level hierarchical organization comprising a bundle ofintertwined groups.
 20. The fabric of claim 1, further comprising a yarnhaving a three-level hierarchical organization, said three-levelhierarchical organization comprising a strand of intertwined bundles.21. The fabric of claim 1, further comprising a yarn having a four-levelhierarchical organization, said four-level hierarchical organizationcomprising a cord of intertwined strands.
 22. The fabric of claim 1,wherein the yarn is twisted at or below 30 twists per inch.
 23. Thefabric of claim 1, wherein a plurality of the yarns are intertwined toform a fabric.
 24. The fabric as recited in claim 1, wherein the fabriccomprises a composite of the sericin-extracted fibroin fibers and one ormore degradable polymers selected from group consisting of Collagens,Polylactic acid or its copolymers, Polyglycolic acid or its copolymers,Polyanhydrides, Elastin, Glycosamino glycans, and Polysaccharides. 25.The fabric of claim 20, wherein a plurality of yarns are non-randomlyorganized into a fabric selected from the group consisting of, wovenfabrics, knit fabrics, warp knit fabrics, bonded fabrics, coatedfabrics, dobby fabrics, laminated fabrics, mesh and combinationsthereof.
 26. The fabric of claim 20, wherein a plurality of yarns arerandomly organized into a non-woven fabric.
 27. The fabric of claim 1,further comprising a drug associated with the fabric.
 28. The fabric ofclaim 1, further comprising a cell-attachment factor associated with thefabric.
 29. The fabric of claim 28, wherein the cell-attachment factoris RGD.
 30. The fabric of claim 1, wherein the fabric is treated withgas plasma.
 31. The fabric of claim 1, further comprising biologicalcells seeded onto the fabric.
 32. A method for forming a fabriccomprising: a. aligning fibroin fibers in parallel or intertwined withother fibroin fibers to form a yarn, b. substantially removing sericinfrom the fibroin fibers without substantially altering the nativestructure of fibroin in the fibers, c. and organizing a plurality ofyarns to form a fabric.
 33. The method of claim 32, further comprisingintertwining the parallel silk fibers before the sericin is extracted.34. The method of claim 32, further comprising intertwining the parallelsilk fibers after the sericin is extracted.
 35. The method of claim 32,further comprising aligning multiple fibroin fibers into yarns, whereineach yarn comprises at least three parallel or intertwined fibers. 36.The method of claim 35, wherein the fibroin fibers of each yarn aretwisted about each other at 0 to 11.8 twists per cm.
 37. The method ofclaim 32, wherein multiple yarns are twisted about each other at 0 to11.8 twists per cm.
 38. The method of claim 32, wherein sericin isextracted from no more than about 50 parallel or intertwined fibroinfibers.
 39. The method of claim 32, wherein the yarn is twisted at orbelow 30 twists per inch.
 40. The method of claim 32, further comprisingforming a knit or woven fabric from a plurality of non-randomlyorganized yarns.
 41. The method of claim 32, further comprising forminga non-woven fabric from a plurality of randomly organized yarns.
 42. Themethod of claims 40 and 41, wherein the fabric is formed after sericinis extracted from the fibers in the yarns.
 43. The method of claims 40and 41, wherein the fabric is formed before sericin is extracted fromthe fibers in the yarns.
 44. The method of claims 40 and 41, wherein theyarn is exposed to a force no greater than its yield point.
 45. Themethod of claim 32, further comprising associating a drug with thefabric.
 46. The method of claim 32, further comprising associating acell-attachment factor with the fabric.
 47. The method of claim 46,further comprising associating RGD with the fabric.
 48. The method ofclaim 32, further comprising treating the fabric with gas plasma. 49.The method of claim 32, further comprising: sterilizing the fabric.